Are mitochondrial genes decoded in the same way as nuclear genes?

Mammalian mitochondrial genomes contain only 22 tRNA-coding genes, which is an insufficient number to decode mRNAs under the standard wobble rules.

How is translation of mitochondrial mRNAs achieved with this number of tRNAs?

The possibilities that occur to me are that certain genes encode multiple tRNAs (but this appears unlikely as each gene has its own promoter) or that mitochondrial tRNAs can adopt alternative three-dimensional structures, each with a different anticodon loop and hence a different anticodon.

Answer Summary

Translation in the mitochondria of mammals differs markedly from that in the cytoplasm. Although it has more similarity to translation in prokaryotes, it also shows significant changes from the latter. A smaller repertoire of tRNAs is able to translate a 'simplified' genetic code through structural alterations and base modifications that allow their anticodons to decode groups of synonymous codons, not possible according to the standard wobble 'rules'.

Relationship to the eubacterial translation system

One of the pieces of evidence for the endosymbiotic theory of the origin of mitochondria was the fact that their translation machinery was more similar to that of prokaryotes than to that in the eukaryotic cytoplasm. The similarities to prokaryotes included the (smaller) size of their ribosomes, the susceptibility of their ribosomes to antibiotics specific for bacterial ribosomes, and the formylation of their initiator tRNA - fmet-tRNA.

Although in no way rebutting the endosymbiotic theory (which is widely accepted), mitochondrial translation shows several differences from that in prokaryotes. These include there being a single tRNA to decode methionine in both initiation and elongation (discussed below), the lack of leader sequences preceding the initiation codon, hence precluding a Shine & Dalgarno type of recognition by the ribosome, and additional ribosomal proteins which are thought to “provide a specialized platform for the synthesis and membrane insertion of the highly hydrophobic protein components of the respiratory chain”.

Decoding with 22 mitochondrial tRNAs

General Review: Suzuki et al. (2011) Annual Review of Genetics 45, 299-329

As the question states, mammalian mitochondria encode 22 mitochondrial tRNAs, which would be insufficient to decode mRNAs using the standard genetic code and the wobble 'rules' that apply to translation in the eukaryotic cytoplasm and in prokaryotes.

One way out of the dilemma would be if some of the tRNAs were encoded in the nucleus and imported into the mitochondrion in a similar manner to that of nuclear-encoded mitochondrial proteins. Although this turns out to be true for certain organisms (although the import system is different from that for proteins), and important in some cases, this is not the answer for mammalian mitochondria. Thus, as reviewed in Current Genetics (2009), the mitochondrial tRNAs encoded in the nuclear genome of H.sapiens only duplicate the 22 mitochondrial tRNAs and their anticodons. (The one exception is an extra Gln(anticodon CUG) in addition to the Gln(anticodon UUG).)

Let us look more carefully at the problem. Although there are 61 codons specifying amino acids, because of wobble, fewer than this number of tRNAs are required to decode them. The left-hand frame of the figure below shows the standard genetic code, and the minimum number of tRNAs needed to decode it using the wobble 'rules' shown beneath, indicated by the colour-coded rectangles.

For those not familiar with the actual pattern of wobble (summarized in my answer to another question), in elongator tRNAs the 5'-position of the anticodon has some flexibility such that a G in that position can form base pairs with U or C in the 3'-position of the codon, an inosine (I) can pair with U,C or A, but an anticodon C can only pair with a codon G. The unmodified bases A and U are rarely found in anticodons, but a variety of different chemically modified Us are found (U* in the figure) which only pair with A. Thus, the minimum number of elongator tRNAs needed would be the number of coloured rectangles - 36 - and, as there are separate species of tRNAmet for initiation and elongation, the minimum number of tRNA species is 37.

When the human mitochondrial genome was sequenced, two anomalies stood together: the limited number of tRNA genes already mentioned and (more sensationally) changes from the standard genetic code (indicated in red on the right-hand side of the figure). It was immediately realized that these changes served to rationalize the code so that all synonymous codons were in groups of four or two. If it were assumed that the decoding specificity or wobble of mitochondrial tRNAs were different, then 22 tRNAs would be sufficient using the 'wobble' shown beneath the right-hand frame of the figure for groups of four amino acids (dark blue) and groups of two amino acids with codons ending in A or G (red).

One further requirement was that a single tRNAmet be involved in both elongation and initiation, a requirement the fulfilment of which has already been mentioned.

What features of the tRNAs could change the wobble 'rules' that have been conserved between prokaryotes and eukaryotic cytoplasm? There turn out to be two types of changes. The first, and most obvious, is the fact that certain (but not all) mitochondrial tRNAs have non-canonical cloverleaf structures, the most extreme being tRNAser in which the whole D-loop is missing. The second is the post-transcriptional modification of bases in the anticodon - just as is the case for tRNAs in the cytoplasm, this can markedly affect decoding specificity.

It is not yet possible to describe the codon-anticodon interaction in mitochondrial translation in the way that this has been done in for the cytoplasm or for prokaryotes. The problem would appear to be the difficulty of obtaining sufficient pure tRNAs. Whereas it is easy enough to clone and transcribe the tRNA genes, this is insufficient without the secondary chemical modifications of the bases which is key to the structure and function of the tRNAs.

It turns out (and I'm not a 100% sure of this) that every tRNA-coding gene could give multiple variants of anticodons by different folding ways, so that you'd have enough anticodons in the end.

New DNA from a Neanderthal bone reveals evidence of a lost tribe of humans

A femur discovered in a cave in southwestern Germany has provided researchers with firm evidence that a small population of humans left Africa and then vanished, long before the big migration that saw humans populate the globe.

Signs of this mysterious early migration remained in the DNA of the Neanderthal who left the leg bone behind, revealing not only a previous tryst between the two hominin populations, but a sign that Neanderthals were far more diverse than we thought.

A team of scientists led by the Max Planck Institute for the Science of Human History and the University of Tübingen in Germany used the DNA from the femur's mitochondria to determine its relationship with other Neanderthals and modern humans.

Neanderthal and human history is a little complicated. So stick with us.

Neanderthals and humans are regarded as close cousins, either under the same species of Homo sapiens or a closely related species Homo neanderthalis.

Mitochondria — our cells batteries — contain a set of genes separate from the DNA bunched up inside our nucleus. Since mitochondrial DNA mutates in a fairly predictable, conserved fashion, we can measure and map its mutations to get a good idea of when two populations last shared them.

Differences between our mitochondrial genes suggest we last shared a common ancestor a little over 400,000 years ago, though previous studies on nuclear DNA had estimated a split as far back as nearly 800,000 years ago.

Another group of human cousins dubbed the Denisovans also split off from a group of Neanderthals roughly 400,000 to 450,000 years ago before they went wandering the Earth.

The thing to note is Denisovans have nuclear DNA that matches Neanderthals' DNA more than our own. Which makes sense, since Denisovans probably split off from a Neanderthal population.

But Neanderthals and modern humans have more similar mitochondria. Why the difference?

Neanderthal bones found in a Spanish cave have been dated to 430,000 years ago, suggesting their ancestors left Africa nearly half a million years ago and ventured across Europe as far as southern Siberia before dying out only a few tens of thousands of years ago.

Our own ancestors migrated out of Africa some time roughly 50,000 years ago, before establishing ourselves across the globe.

DNA taken from modern humans with non-African lineage reveals we have genes that had evolved in Neanderthals and Denisovans, suggesting there was a bit of an on-again/off-again relationship with our cousins over the millennia since we first parted ways.

Considering the populations had a chance to mingle in Europe over a span of a few thousand years, some sort of casual affair isn't all that surprising.

But this new discovery is a bit of a shock.

The specimen, coded HST after the site of its discovery in Hohlenstein-Stadel cave, couldn't be carbon-dated. But its mitochondrial DNA put it at about 124,000 years old.

"The bone, which shows evidence of being gnawed on by a large carnivore, provided mitochondrial genetic data that showed it belongs to the Neanderthal branch," says lead researcher Cosimo Posth of the Max Planck Institute for the Science of Human History.

Just to throw another twist into the story, this Neanderthal's mitochondria didn't come from the same group as those belonging to other previously analysed Neanderthal bones. Instead, it came from a lineage dating back at least 220,000 years.

Not only does this suggest modern humans might have been stepping tentatively into Europe and getting friendly with Neanderthals long before the wave of migration that led to today's population, it shows Neanderthals were more diverse than we thought.

Taken altogether, this evidence helps flesh out the complex relationship between Neanderthals, Denisovans, and modern humans.

Around 450,000 years ago, an ancestor of the Neanderthals and Denisovans split off and headed for Europe and Asia. Those who ventured further east eventually became the Denisovans in the west, they were the Neanderthals.

Around 200,000 years later, a small group from our own ancestral line ventured out of Africa and bred with Neanderthals. This now lost tribe of humans was large enough to leave their mitochondria, but not so big to leave a significant mark on the Neanderthal's nuclear DNA.

"This scenario reconciles the discrepancy in the nuclear DNA and mitochondrial DNA phylogenies of archaic hominins and the inconsistency of the modern human-Neanderthal population split time estimated from nuclear DNA and mitochondrial DNA," says researcher Johannes Krause, also of the Max Planck Institute for the Science of Human History.

This picture is just one possible explanation. It's also possible that the bone comes from another distinct population that had itself migrated from Africa.

With the recent discovery of anatomically modern humans evolving 100,000 years earlier than previously estimated, it's not out of the question that our ancestors did a lot of moving about.

Further discoveries could shine more of a light on the interactions between our human ancestors. Until then, the relationship status between Neanderthals and humans is 'it's complicated'.


De-Hui Yuan 1† , Jian-Feng Xing 2† , Mei-Wei Luan 2 , Kai-Kai Ji 2 , Jun Guo 2 , Shang-Qian Xie 2* and Yuan-Ming Zhang 1*
  • 1 Crop Information Center, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
  • 2 Key Laboratory of Genetics and Germplasm Innovation of Tropical Special Forest Trees and Ornamental Plants (Ministry of Education), Hainan Key Laboratory for Biology of Tropical Ornamental Plant Germplasm, College of Forestry, Hainan University, Haikou, China

DNA 6mA modification, an important newly discovered epigenetic mark, plays a crucial role in organisms and has been attracting more and more attention in recent years. The soybean is economically the most important bean in the world, providing vegetable protein for millions of people. However, the distribution pattern and function of 6mA in soybean are still unknown. In this study, we decoded 6mA modification to single-nucleotide resolution in wild and cultivated soybeans, and compared the 6mA differences between cytoplasmic and nuclear genomes and between wild and cultivated soybeans. The motif of 6mA in the nuclear genome was conserved across the two kinds of soybeans, and ANHGA was the most dominant motif in wild and cultivated soybeans. Genes with 6mA modification in the nucleus had higher expression than those without modification. Interestingly, 6mA distribution patterns in cytoplasm for each soybean were significantly different from those in nucleus, which was reported for the first time in soybean. Our research provides a new insight in the deep analysis of cytoplasmic genomic DNA modification in plants.

OK, we’re part Neanderthal, and not that much different from chimpanzees after all. We also know that some drugs won’t work on my cancer, even though they might work on yours.

And, if you want to find out what your DNA has been saying behind your back, the price of having your personal genome decoded is dropping like a stone.

The map of the human genome, completed in 2001, has wowed scientists in the years since, even if the scale of its impact has not matched some of the early predictions surrounding the project.

Eric Lander, a leader of the Human Genome Project, said Tuesday (Feb. 22) that he has been surprised at the pace of advances stemming from the project, which has been likened to “biology’s moon-shot.”

“This has gone so much faster than I ever imagined,” said Lander, president and director of the Broad Institute of Harvard and MIT and professor of systems biology at Harvard Medical School. “We’ve been able to read out the lab notebooks of evolution.”

Lander was part of a panel of scientific experts who talked about the Human Genome Project and its legacy at Sanders Theatre.

The event, “Mapping the Human Genome: Ten Years After,” was hosted by Harvard President Drew Faust and webcast by USA Today. Other panelists were Margaret Hamburg, head of the U.S. Food and Drug Administration M. Susan Lindee, chair and professor of history and sociology of science at the University of Pennsylvania Vamsi Mootha, associate professor of systems biology and of medicine at Harvard Medical School and associate member of the Broad Institute and Vicki Sato, former president of Vertex Pharmaceuticals and today professor of the practice of molecular and cellular biology in the Faculty of Arts and Sciences and professor of management practice at Harvard Business School.

“Has sequencing of the human genome been transformative, and in what ways?” Faust asked in her introductory remarks. “How are we all … different than we otherwise would have been and what will the coming decades hold?”

The Human Genome Project started in 1990 and involved scientists from 20 centers in six countries. At a cost of $3 billion, the first draft of the genome was published in 2001 and the full sequence was published in 2003. The resulting map shows 21,000 genes that provide the instructions for making a human being and provide the foundation for better understanding our basic biology, how we differ from other animals, and what happens when things go wrong.

Lander reeled off a list of advancements made possible by the project. In barely the time it takes to get a single drug developed and approved for use in humans, the number of genes tied to common diseases such as diabetes and Alzheimer’s has increased from just 20 to 1,100, with more on the way. Scientists have decoded the genomes of dogs, chimpanzees, laboratory animals, and a host of other creatures. The cost of reading a genome has fallen dramatically in just 10 years. Technology today can do in five minutes a decoding task that would have taken a year to complete a decade ago, Lander said.

The revolution extends to students and young researchers, who have at their disposal not only a new understanding of the foundation of life, but an array of equipment and techniques that didn’t exist not long ago.

“They have the tools to do things today that it used to take armies to do,” Lander said. “My expectations have been blown away.”

When it comes to the public’s expectations, however, hype surrounding the project may have led some to imagine an era of rapidly developed genetic cures, Sato said.

Instead, as our understanding has advanced, the complexity of many diseases has emerged.

But that’s not to say the Human Genome Project hasn’t had an effect on some illnesses, Mootha said.

“I’m really excited about the future. If I were a student or graduate student, I don’t think there’s a better time to embark on biomedical research.”

One problem, Mootha said, is that there is a “tsunami” of data from all the genetic analysis going on, so much so that equipment can’t handle or even store it.

Another problem is the nature of the information, Lindee said. Though people are getting more information about their genetic tendencies, some of the information is ambiguous and difficult for doctors and patients to interpret, leading to an increase in what she called “uninformative information.”

Sato said it has taken time for pharmaceutical companies to change how they operate to take advantage of a flood of genomic data. They have had to change the way they look at disease, but they also have gained a better understanding of the differences between patients, and the fact that certain medicines work better on some people than others.

“If they have a mutation, we know this drug won’t work even though it is the same cancer [as another person without the mutation],” Sato said. “So much has changed in a relatively short period of time, I can’t imagine what the medicine of the future will look like.”

Data Records

Raw reads from Illumina are deposited in the NCBI Sequence Read Archive (SRA) 57,58,59,60,61,62 and BIG Genome Warehouse 63 . Assembled cp genome sequences and accompanying gene annotations of C. sinensis var. assamica are deposited in the NCBI GenBank 64 and BIG Genome Warehouse 65 . The mt genome final assembly and accompanying gene annotations are deposited at NCBI GenBank 66,67 and BIG Genome Warehouse 68 . The alignment and tree files of the chloroplast genome and mitochondrial genome form the Camellia genus were deposited in Figshare database 69 .

Almost 20 years of Neanderthal palaeogenetics: adaptation, admixture, diversity, demography and extinction

Nearly two decades since the first retrieval of Neanderthal DNA, recent advances in next-generation sequencing technologies have allowed the generation of high-coverage genomes from two archaic hominins, a Neanderthal and a Denisovan, as well as a complete mitochondrial genome from remains which probably represent early members of the Neanderthal lineage. This genomic information, coupled with diversity exome data from several Neanderthal specimens is shedding new light on evolutionary processes such as the genetic basis of Neanderthal and modern human-specific adaptations—including morphological and behavioural traits—as well as the extent and nature of the admixture events between them. An emerging picture is that Neanderthals had a long-term small population size, lived in small and isolated groups and probably practised inbreeding at times. Deleterious genetic effects associated with these demographic factors could have played a role in their extinction. The analysis of DNA from further remains making use of new large-scale hybridization-capture-based methods as well as of new approaches to discriminate contaminant DNA sequences will provide genetic information in spatial and temporal scales that could help clarify the Neanderthal's—and our very own—evolutionary history.

1. Introduction

The best way to understand our evolutionary history as modern humans is comparing our own genome with those of our closest relatives. The genetic bases of the traits that we do not share with them are going to be those that define our singularity as a species. Until recently, we only had the chimpanzees for such comparisons however, our lineage and that leading to them probably separated more than 6 million years ago and thus, they constitute a very distant relative. Let us take language for instance, our unique ability to communicate abstract ideas that is often inferred to set us apart from the rest of the natural world. Chimpanzees do not speak, not only because they have a different brain and a different genetic make-up, but also because they do not have the vocal tract that enables us to produce the sounds we use for it. Therefore, it is quite clear that, for understanding adaptive processes that probably took place not at the origin of the hominin lineage, but millions of years afterwards, the chimpanzee represents a rather poor reference.

Depending on which adaptive processes are addressed, an obvious source of comparison would be to obtain genetic data from fossils that represent remains of our hominin relatives. Given that Neanderthals are our closest and best-known relatives, in addition to their prevalence up to the late Pleistocene (giving more chances for DNA preservation), this makes them ideal candidates to identify those traits that might have originated within our own evolutionary lineage.

2. Neanderthal mitochondrial DNA sequences

The first Neanderthal sequence was obtained in 1997 by a team led by Svante Pääbo. They were able to recover the mitochondrial DNA (mtDNA) hypervariable region 1 by polymerase chain reaction (PCR) from the Neanderthal holotype specimen from Feldhofer cave in Germany. By comparing it against a panel of worldwide present-day human mtDNA sequences, the data indicated that Neanderthals were a sister group to anatomically modern humans, providing no evidence of interbreeding between Neanderthals and modern humans, at least to a level sufficient to result in Neanderthal mtDNA introgression into the modern human mtDNA gene pool [1].

During the 15 years following this publication, other Neanderthal sequences from different sites such as Mezmaiskaya (Russia) and Vindija (Croatia) in 2000, Engis (Belgium), La Chapelle-aux-Saints (France), Les Rochers-de-Villeneuve (France) in 2004, El Sidrón (Spain) in 2005, 2006 and 2011, Monti Lessini (Italy) and Scladina (Belgium) in 2006, Teshik-Tash (Uzbekistan) and Okladnivok (Russia) in 2007 and Valdegoba (Spain) in 2012 were successfully amplified with the same technical approach [2–12] (figure 1). A common observation of all these studies was that Neanderthal mtDNA sequences were similar to each other—suggesting a general low diversity—and different to any reported modern human mtDNA. Furthermore, some studies began analysing a possible phylogeographic structure the basal sequences in the phylogenetic trees were from the easternmost Neanderthals (located in Central Asia) or from the oldest ones (Valdegoba and Scladina) [12]. This seems to support an east–west genetic cline and also the existence of temporal bottlenecks that shaped the mtDNA diversity. Recent western European Neanderthals (roughly less than 50 000 years) constitute a tightly defined group with low mitochondrial genetic variation in comparison with both eastern and older (more than 50 000 years) European Neanderthals. Eastern and western Neanderthals seem to have diverged approximately 55 000–70 000 years ago followed by an extinction of western Neanderthals throughout most of their range and a subsequent recolonization of the region [12].

Figure 1. Geographical map showing Neanderthal and Denisovan sites with different types of genetic data (partial mitochondrial, complete mitogenomes, exomes, partial nuclear data or complete genomes) retrieved.

However, to explore these migration patterns across time and space, we need to have a basic understanding of the Neanderthals' demography. Fortunately, there is a Neanderthal site that can provide such information because it may represent a family group. The Spanish site of El Sidrón is thought to be a synchronic accumulation of at least 12 Neanderthals including three female and three male adults, three adolescents, two juveniles and one infant. Complete and partial mtDNA sequences from all the available individuals suggest that Neanderthals there formed small kinship-structured bands that practised patrilocal mating behaviour and had relatively long inter-birth intervals (ca 3 years) when compared with modern human populations. In addition to providing intriguing anthropological insights into a Neanderthal social group—similar features have also been described in modern hunter–gatherers—such information may help in choosing demographic parameters when generating models of Neanderthal population dynamics [1].

3. Mitochondrial genomes and the advent of the new sequencing technologies

With the introduction of next-generation sequencing (NGS) technologies to the field of ancient DNA (aDNA), it was possible for the first time to retrieve complete mitochondrial genomes, first by shotgun sequencing of a sample from Vindija cave [13] and later with targeted hybridization-capture enrichment methods [14]. The whole mtDNA genome allowed a more precise estimate of the divergence time between modern human and Neanderthal mtDNA lineages, which was reported to be 660 000 years considering all sites of the mtDNA [13] or close to 400 000 years considering only third codon sites of the mtDNA [15]. Another striking observation was that the ratio of non-synonymous to synonymous evolutionary rates was significantly higher on the Neanderthal lineage, a result that would fit with Neanderthals having a smaller effective population size, and thus evolving under lower selective constraints than modern humans [13]. By 2009, the analysis of six complete Neanderthal mtDNA genomes indicated that the variation among Neanderthals was approximately one-third of that estimated for present-day humans worldwide, suggesting a female effective population size of less than 3500 individuals [14]. This finding was surprising given that the Neanderthal sequences stem from several distinct time points spanning thousands of years across a wide geographical range, and thus it appears to be a conservative estimate with respect to sampling at a contemporaneous time period. The most recent common ancestor (MRCA) of the Neanderthal samples analysed was estimated to have lived approximately 110 000 years ago, which is much less than the age estimated for modern human mtDNAs [3].

Furthermore, these new sequencing technologies allowed precise estimates of modern human contamination in the high-coverage mtDNA genomes obtained, but also the description of misincorporation patterns related to cytosine deaminations at the edge of the sequencing reads that is characteristic of aDNA sequences, and increases with time [16,17]. In subsequent studies, these patterns allowed the identification of authentic Neanderthal sequences and opened up the possibility of analysing Neanderthal samples that were previously discarded for genetic studies due to their high level of present-day human contamination [18].

4. The first nuclear DNA sequences

As Neanderthal mitochondrial diversity was being studied, attention also turned to nuclear loci. Although challenging, given the lower proportion of nuclear DNA compared with mtDNA, researchers were thrilled by the idea as it unlocked the possibility of assessing whether emblematic functional and phenotypic modern human traits were shared by Neanderthals.

Between 2007 and 2009, by amplifying small nuclear regions encompassing functional variants, researchers found that some Neanderthals were probably red-haired and pale skinned [19], they had bitter taste perception ability [20] and presented the ABO blood type O [21]. In addition, having the same functional variants as modern humans in the FOXP2—a gene that when mutated generates a speech and language impediment—suggested that Neanderthals might have been able to communicate with similar language capabilities to ours, or at least they had the genetic basis to do so [22]. Nonetheless, recent studies found differences between most modern humans and Neanderthals in a regulatory element near the FOXP2 gene that could have functional implications [23].

While recovering short pieces of nuclear DNA became possible in well-preserved and uncontaminated specimens, the sequencing of a whole Neanderthal genome remained a difficult challenge, owing to the low amount of nuclear DNA sequences relative to environmental sequences, and the limitations of the available technology. Two pioneer studies managed to recover 65 kb of nuclear DNA and 1 Mb of sequence of Neanderthal nuclear DNA by cloning and sequencing short fragments of DNA [24] or by metagenomic sequencing [25], respectively. They estimated coalescence times between modern humans and Neanderthals to be roughly between 700 000 and 500 000 years ago. However, it was subsequently demonstrated that a significant fraction of the data generated by the second study derived from modern human contaminant DNA [26]. As a result of this early pitfall, more stringent measures were taken while constructing the sequencing libraries, eliminating potential environmental and modern human contamination [27,28].

5. The Neanderthal and Denisovan draft genomes

The year 2010 saw not only the publication of the long-expected Neanderthal draft genome [28] but also that of a previously unknown hominin, called Denisovan, named after the cave in the Altai Mountains where the remains were discovered [29]. Currently only two teeth and a finger bone (the latter with extraordinary levels of DNA preservation, approx. 70% of endogenous DNA) have been attributed to the Denisovans. Both nuclear and mtDNA extracted from these remains suggest that Denisovans were as genetically diverse as two present-day humans from different continents and more diverse than Neanderthals from throughout their range, suggesting that their effective population size was relatively large [30] (see also a later discussion in [31]). By employing a user-defined hybridization-capture method, a high-coverage mtDNA genome from the Denisovan finger bone was retrieved [32], and it was estimated that it diverged from the common ancestor of modern humans and Neanderthals around 1 million years ago [33]. Moreover, as both nuclear archaic genomes were sequenced, clearer phylogenetic relationships were established for the first time. The MRCA of modern humans, Neanderthals and Denisovans was found to have lived at least 800 000 years ago, whereas the Denisovan and Neanderthal genomes were more closely related to each other—as sister species—and their divergence time was around 600 000 years ago.

In addition to the general hominin phylogeny, the analysis of five present-day humans from different continental areas suggested that non-Africans shared 1–4% more derived alleles with Neanderthals than with sub-Saharan Africans [28], whereas present-day Melanesians also seemed to share 4–6% of their DNA with the Denisovan individual. The Neanderthal signal was later also observed in African populations, which is likely the result of back-to-Africa migrations [34–36]. These results were interpreted as evidence of Neanderthals interbreeding with the ancestors of all non-Africans and subsequently a Denisovan-like population mainly with the ancestors of South East Asians [37] however, marginal Denisovan admixture has also been reported in continental Asian populations [31,38], further entangling this later admixture scenario. This notwithstanding, the proportions of admixture are probably overestimates if some degree of structure was present among ancient humans in Africa, as already pointed out in [28,39–41]. If this were the case, incomplete lineage sorting and not introgression could explain some genetic similarities between modern non-African humans and Neanderthals, although certainly not all of them.

6. High-coverage genomes

A major technical breakthrough in 2012 involved a novel library preparation method that exploited single-stranded DNA and greatly increased the yield of sequencing from ancient samples. Briefly, instead of building the libraries exclusively from double-stranded DNA—where only sequences without ‘nicks' or single-strand breaks can be incorporated into NGS libraries—the new method first denatures DNA fragments and incorporates the single strands of DNA into NGS libraries, allowing for the recovery of significantly more DNA molecules than hitherto possible. By applying this new method, a 30X coverage genome from the same Denisovan sample [42] and a 54X coverage genome from a female Neanderthal toe bone [31] also from Denisova Cave—known as the Altai Neanderthal—were generated.

Having high-quality genome data not only offers refined insights into Neanderthal relatedness to modern humans, but also allows us to start addressing questions concerning their diversity and demographic history, something that could not be done with low coverage data. For instance, under a no gene flow scenario, the date of the split of the archaic and modern human populations, which by necessity is more recent than sequence divergence, can be estimated. Recently, mutation rates have also been a subject of debate [43]. Based on a mutation rate of 1.03 × 10 −9 derived from the fossil record (which is essentially two times faster than the genealogical one), the population split between Denisovans, Neanderthals and modern humans probably occured between 383 000 and 257 000 years ago, whereas the populations that evolved into Neanderthals and Denisovans separated roughly 236 000–190 000 years ago [31].

A more precise idea of how and when the admixture with archaic humans occured is also beginning to emerge. By coupling high-coverage archaic and present-day human genomes, the amount of DNA introgressed from Neanderthals into non-sub-Saharan Africans has been refined to a range of 1.5–2.1% of Neanderthal ancestry in present-day populations [44]. It has also been observed that Neanderthal-derived DNA in all non-Africans is more closely related to a low coverage genome from the Mezmaiskaya skeleton in the Caucasus than to the Altai or to the Vindija genome [31]. The linkage disequilibrium pattern of haplotypes of suspected Neanderthal origin suggests a date of admixture between 37 000 and 82 000 years ago [45]. Altogether, these observations seemed to indicate that a currently unsampled Middle Palaeolithic Neanderthal population living in the Levant and/or western Asia encountered modern humans as they migrated out of Africa, subsequently spreading the signature of introgression as they populated the rest of the world.

Furthermore, it has recently been shown that East Asians and native Americans may have between 1.7 and 2% more Neanderthal admixture than other non-African populations, which suggests that a second introgression event took place after European and Asians populations diverged [42,46]. This latter finding was unexpected given the archaeological evidence of a long-term occupation of Neanderthals in Europe and a possible late overlap with early modern human migrations into Europe. Moreover, Late Palaeolithic and Mesolithic modern human genomes have so far failed to demonstrate a closer relatedness to Neanderthals [47,48]. High-coverage genomes of Late Pleistocene Europeans—and also from other populations—will be needed to estimate accurately if other admixture events could have occured with Neanderthals or Denisovans. Interestingly, some lines of evidence suggest that interbreeding may have been limited by genetic incompatibilities (below) and thus a short-lived increase in Neanderthal admixture would only be observed close to the interbreeding events(s) [44].

In addition to determining the phylogenetic relationships among hominins, a potentially interesting application of the high-coverage genomes is to investigate in detail the introgressed regions and see whether they harbour genetic variants that could be beneficial to modern humans. Several recent publications suggest that some archaic variants could have been advantageous or at least functionally relevant after being introgressed into modern humans [49–53]. For instance, Neanderthal haplotypes in European and East Asians are enriched for genes harbouring keratin filaments—a protein expressed in skin, hair and nails—suggesting that skin or hair adaptation to non-African environments was enhanced after the admixture event [53]. Inversely, there seem to be large ‘deserts' of Neanderthal ancestry, which implies that selection may have acted to remove genetic material derived from Neanderthals [44,54]. Furthermore, genes that are more highly expressed in testes than in any other tissue are especially reduced in Neanderthal ancestry, and there is an approximately fivefold reduction of Neanderthal ancestry on the X chromosome [44] these observations can be interpreted as selection eliminating Neandertal-derived genes that may have reduced male fertility. Furthermore, the known differences in effective population size between East Asians and Europeans could have resulted in less efficient selection to remove Neanderthal-derived deleterious alleles and thus be the cause for the excess of Neanderthal signal observed in East Asians populations [44], although others suggested it was more probably attributable to further interbreeding in the East [54], as suggested earlier.

7. Neanderthal genomic diversity and demographic trends

The opportunity to analyse large genomic regions from different Neanderthal specimens opens the possibility of studying diversity patterns that could be related to specific demographic and evolutionary processes, and that can also shed light on their extinction process.

The recent advent of the high-coverage exomes of two Neanderthals, one from Vindija 33.15 (40X) in Croatia and the other from El Sidrón SD1253 in Spain (12X) [55] (figure 1), has allowed a start in addressing those subjects. Together with the exome regions of the Altai and the Denisovan genomes, the Neandertal exomes have been compared with the same regions from three modern individuals from Africa, Europe and Asia/Pacific. Interestingly, it was found that the average heterozygosity—the number of nucleotide differences within an individual per thousand base pairs—among the three Neanderthals was 0.128, which is approximately a third of what is seen in present-day humans. The three Neanderthals have longer runs of homozygosity than modern humans. The Altai individual has been reported to have an inbreeding coefficient of one-eighth—indicating that the parents were as closely related as half-siblings. Additionally, possible weaker consanguinity signals are also present in the Vindija and El Sidrón material. Additional samples would be of paramount importance to see whether the homozygosity tracks increase in length over time, and whether this correlates with the extinction process. Considering the two individuals securely dated (approx. 44 000 years ago for Vindija 33.15 and approx. 49 000 years ago for El Sidrón 1253), the homozygosity tracks longer than 200 kb almost double in about 5000 years [55]. In addition, the genetic differentiation among individuals is larger among Neanderthals than among present-day humans. This suggests that Neanderthals lived in small and relatively isolated populations, which probably caused them to become more differentiated from each other when compared with modern humans.

Furthermore, inferences from the high-coverage Neanderthal and Denisovan genomes [31] suggest that some time after 0.5–1.0 million years ago their ancestral populations decreased in size for hundreds of thousands of years. A low population size over a long time would reduce the efficacy of purifying selection and contribute to a larger fraction of likely deleterious alleles, particularly at low frequency. In accordance with what would be expected of a long-term low population size, the Neanderthal exomes show that the proportion of all derived SNPs that are inferred to change amino acids and to be deleterious—assessed from alleles expected to affect the protein function or that occured in conserved positions—is larger than in modern human populations. Among derived amino acid-changing alleles likely to be at low frequency in Neanderthals, not only a higher proportion is inferred to alter protein function, but also they seem to be the functional variants with the most deleterious consequences when compared with SNPs at lower frequency in the modern human populations. However, it is interesting to note that these results seem not to affect the deleterious load per individual, since the number of genes associated with non-dominant traits with heterozygous- or homozygous-derived alleles inferred to be deleterious, is not different between Neanderthal and present-day individuals [56]. Therefore, susceptibility of Neanderthals to any specific genetic disorder cannot be inferred from these data [55].

8. Modern human- and Neanderthal-specific traits

The high-coverage Neanderthal and Denisova genomes now provide a sound basis to identify genomic changes specific to modern humans and, with that, a list of substitutions accountable for ‘what makes us modern humans' has emerged [30,31,38].

Moreover, the exomes of the three Neanderthals and the Denisovan individual allow us, for the first time, to identify derived amino acid changes shared by three Neanderthals as well as the Denisovan individual that are not seen, or only occur at a very low frequency, in present-day humans. Such changes are of interest since they may underlie phenotypes specific to the archaic populations. By calculating the fraction of all amino acid changes specific to either the archaic or modern human lineages for each phenotype category of genes in the Human Phenotype Ontology database, an estimation of the enrichment of amino acid changes in phenotypes in each archaic lineage has been obtained [55]. The authors find that genes involved in skeletal morphology may have changed more on the Neanderthal and Denisova lineages than on the preceding lineage from the common ancestor shared with chimpanzees. These genetic changes could underlie some skeletal Neandertal traits such as a reduced lordosis—the curvature of the lumbar and cervical spine unfortunately, the fact that there is so far little morphological evidence from Denisovans hinders corroborating further associations between genetic changes and morphological traits in the lineage specific to archaic humans. In the modern human lineage, there is an overrepresentation of some behavioural genes intriguingly, some of these genes have been related to traits such as ‘hyperactivity’ or ‘aggressive behaviour’ [55].

Thus, most of our understanding of the biology of ancient humans will no longer be limited by the inaccessibility of the data but by our functional interpretation of modern human genomes [57]. Moreover, regulatory changes have also been shown to be of importance in recent human evolution [58], and thus not only coding variants should be taken into account when reconstructing the biology of archaic humans from genetic data.

Nevertheless, functional studies will be essential to better understand the function and importance not only of genetic variants already discovered and specific to the modern human lineage, but also the Neanderthal-lineage-specific changes.

A recent study has decoded the ancient methylation patterns from NGS data to infer the gene expression of a Palaeo-Eskimo individual approximately 4000 years old [59]. Moreover, further work [60] suggests that even though archaic and modern humans share more than 99% of their genetic sequence, there seem to be methylation differences between these hominin groups that are twice as likely to occur in genes implicated in disease, especially brain disease-associated regions, than in genes that are not associated with illness. Methylation differences are also found in HOXD, a gene cluster that regulates limb development, suggesting that some of these epigenetic patterns may explain why, for example, Neanderthals had short distal limb segments in comparison with many modern humans. However, in order to assess what the observed epigenetic differences mean in terms of biology, further functional experiments are necessary. Nonetheless, both of these publications suggest that it will be possible to track epigenomic information through time, and thus they have set up the foundations for yet another new discipline: palaeoepigenetics [59,60].

9. Super-archaic DNA

The mtDNA genome of a ca 400 000-year-old hominin from the Sima de los Huesos in Atapuerca (Spain figure 1) has been sequenced recently [61]. Interestingly, the skeletal remains had previously been classified as H. heidelbergensis and dated to approximately 600 000 years ago, but both the classification and the date were the subject of dispute [62], and given that the remains exhibit a number of derived Neanderthal traits they have been postulated as the ancestors of Neanderthals. A recent analysis of 27 individuals from this palaeontological site (now dated to ca 430 000 years ago) shows that these ‘Sima de los Huesos' hominins present many Neanderthal-derived traits in their face and teeth, whereas the braincase still retained ‘primitive’ conditions [63] it seems that late Neanderthal braincase shapes are not found in Europe before approximately 200 000 years ago. Thus, these data suggest that Neanderthal features did not evolve as a block but rather they were fixed at different rates and paces in different parts of the anatomy. Moreover, and further complicating the scenario, the only Sima mtDNA sequence obtained so far seems to be phylogenetically most similar to that of Denisovans [61], found thousands of miles away, and much younger in age. Although nuclear genome sequences of these specimens would be needed to ascertain their precise relationship to archaic and modern humans, this study provides evidence that aDNA techniques have become sensitive enough to recover and analyse DNA from Middle Pleistocene hominin remains, even from non-permafrost environments.

Furthermore, although morphological evidence suggests that Neanderthal features were already present in European fossils over 400 000 years ago, and that by 130 000 years ago their characteristic suite of traits was fully established [64], no genetic information has yet been recovered from samples older than 100 000 years. It seems obvious that many relevant evolutionary processes took place between these two dates, perhaps related to dramatic climatic events and triggered by the action of genetic drift [64]. Moreover, it is not clear yet whether Neanderthals from other geographical areas or time periods are genetically similar to the ones that have already been analysed. While there are clearly differences between early and late members of the Neanderthal lineage, opinions vary over the unity of European and Asian varieties of this hominin group [64,65]. It will be interesting to address how Neanderthals from different time points related to each other and to what extent climatic conditions or other factors contributed to shape their genetic diversity through adaptation and also demographic reductions and expansions.

Furthermore, having Neanderthal serial time data will enable us to move from a primarily descriptive basis of their demographic history and population dynamics to estimate genetic parameters, for instance, their mutation rate, precise temporal population sizes or local diversity patterns.

10. Extinction process

Almost 20 years of Neanderthal palaeogenetics and palaeogenomics have shown us that Neanderthals, an extinct human population, shared a common evolutionary history with modern humans until approximately 0.5 million years ago. Recent studies suggest that after their lineages separated, their demographic trajectories differed: while the Neanderthal population decreased in size for hundreds of thousands of years (as did that of Denisovans), the ancestors of present-day humans stabilized or increased in number [31]. This observation makes sense in the light of what we know about their genetic diversity, which was no more than a third of what has been estimated for modern humans worldwide. In addition, their coding gene patterns show evidence of reduced efficiency of purifying selection and a larger fraction of probably deleterious alleles, particularly at low frequency. Although we are beginning to grasp general patterns of Neanderthal genetic diversity, we cannot completely understand the consequences of their particular demography and population dynamics unless more specimens contemporaneous with each other are analysed. These data will be paramount in helping us understand to what extent Neanderthals were affected by their small population size, relative isolation and inbreeding practices. For example, the new data might allow us to observe whether they displayed a significant accumulation of variants associated with recessive disorders in comparison with modern humans. While this is just a hypothesis, it could be that an accumulation of genetic deleterious effects associated with decreased effective population size, exacerbated by inbreeding practices in the last Neanderthals, may have contributed to their final demise.

11. Future developments

To address some of the previously mentioned unsolved questions about the Neanderthals' evolutionary history, extensive sampling of new fossils will be needed, and even though ongoing archaeological excavations will hopefully continue to produce material for aDNA studies, it is clear that a number of Neanderthal samples of interest may be stored within museums under less than ideal conditions, or may not have been excavated and handled with enough care to prevent contamination [66] (figure 2). Two main caveats arise from this: many specimens will probably have low endogenous DNA contents, and might have been contaminated significantly with modern human DNA.

Figure 2. An anti-contamination protocol developed at El Sidrón Neanderthal site in Spain to properly handle ancient samples for DNA analysis. The specimens are excavated with sterile laboratory gear and immediately frozen.

As samples from older periods are screened in the search for precious genetic material, even sequencing a mitochondrial genome may require significant amounts of bone tissue [61], which may conflict with conservation purposes. Furthermore, target capture techniques have proved to be most efficient in accessing samples with low endogenous DNA however, only certain genomic regions (e.g. mtDNA or exomes) have been retrieved with high-coverage using this approach [32,55,67]. Recently, a whole genome capture method that uses home-made biotinylated RNA probes as bait (which significantly reduces the cost of probe design) has been developed [68]. While this approach sounds attractive, it seems to introduce a bias against shorter DNA molecules, which is something that will have to be addressed before it can be fruitfully applied to samples of very degraded (and therefore short) Neanderthal DNA [69].

Moreover, regardless of whether samples have been recently excavated or handled without proper anti-contamination measures, as older specimens or samples stemming from a large range of latitudes and site-specific conditions are analysed, a significant proportion of present-day human contamination can be expected. At present, contamination is efficiently estimated, but only two in silico approaches have been developed to putatively separate endogenous from contaminant material [18,70]. However, neither of them precludes the sequencing of contaminant material, which might not be suitable if a high number of poorly handled and preserved samples have to be screened.

Even though very well-preserved samples have been found, it is unlikely that we will discover very ancient samples with an elevated content of endogenous DNA. Therefore, new methodological approaches for enriching the amount of endogenous material, by retaining only informative damaged molecules, will need to be developed to make large screenings economically feasible. Nonetheless, aDNA studies will still be limited by the amount of endogenous DNA present in the sample. Until new methodological approaches are available, target capture and even shotgun sequencing will no doubt continue to be used, depending on the nature of the samples and the scientific questions being addressed. However, it remains to be seen whether single molecule sequencing technologies can, efficiently and without error, transform the field of aDNA and hominin palaeogenomics.

Given that most historic Neanderthal samples are of great value to understand key aspects of their population dynamics and biology across time, new experimental and computational methods will be crucial to access the endogenous DNA required to fully explain Neanderthal and our own evolutionary histories.

There is 'alien DNA' everywhere, thanks to gene thieves

What is the world's most resilient and hardy lifeform? The cockroach has a reputation for toughness &ndash many people seem convinced it could even survive a nuclear apocalypse. The tardigrades, or water bears, are probably even hardier. We know for sure that they can survive being blasted into the hostile emptiness of outer space.

Now it is time to meet another contender: a species of algae that thrives in Yellowstone's bubbling hot springs, deep down in the belly of the Earth where the water is as corrosive as battery acid and the environment is laced with arsenic and heavy metals.

The algae's secret? Theft. It stole the genes it needs to survive from other lifeforms. It is a tactic that may be more common than we think.

Most living creatures that live in extreme places are single-celled microbes &ndash bacteria or archaea. These simple and ancient lifeforms lack the more sophisticated biology of animals, but their simplicity is an advantage: it leaves them much better able to cope with extreme conditions.

They have spent billions of years hiding out in the most inhospitable places &ndash deep underground, at the bottom of the ocean, in freezing permafrost or in boiling hot springs. They have taken the long journey, evolving genes over millions or even billions of years that help them cope with almost anything.

But what if other, more complex creatures could just come along and steal those genes? They would effectively take an evolutionary shortcut. In one move they would have the genetics to survive in extreme places. They would get there without putting in the millions of years of evolutionary hard slog it normally takes to evolve those abilities.

This is exactly what the red alga Galdieria sulphuraria has done. It can be found living happily in the hot sulphur springs of Italy, Russia, the US's Yellowstone Park, and Iceland.

Those hot springs have temperatures as high as 56C. Although some bacteria can live in pools at about 100C, and a few can cope with temperatures of about 110C close to deep-sea vents, it is remarkable that a eukaryote &ndash a group of more complex lifeforms that includes animals and plants (red algae is a plant) &ndash can cope with life at 56C.

Most plants and animals could not tolerate those temperatures, and for good reason. Heat causes the chemical bonds within proteins to break, which makes them collapse. This has a catastrophic effect on enzymes, which catalyse the body's chemical reactions. The membranes that encase cells also become leaky, allowing molecules in that would normally be kept out. Once a certain temperature has been reached, the membrane breaks and the cell falls apart.

However, even more impressive is the algae's ability to tolerate acidity. Some of the hot springs have pH values between 0 and 1.

It is positively-charged hydrogen ions, also known as protons, that make something acidic. These charged protons interfere with proteins and enzymes inside cells, messing up the chemical reactions vital for life.

Rather than inheriting its superpowers from its ancestors, the algae stole them from bacteria

That is because proteins are held together by the mutual attraction between positive and negatively charged amino acids. When you introduce a whole new load of positively charged particles you upset the careful balance holding the protein together. The protein can no longer maintain its specific shape and can therefore no longer do its job properly.

"Most other lifeforms can't withstand extreme heat or acidity," says Gerald Schoenknecht, a plant biologist at Oklahoma State University in Stillwater. "Galdieria survives pH 0, which is equivalent to surviving in dilute battery acid. Most other organisms, even bacteria, cannot handle pH values that low."

However, it is not just heat and acidity that Galdieria can tolerate. The alga is resistant to arsenic, mercury and can live in extremely salty environments. The poisonous elements are usually deadly to life, as they inhibit important enzymes involved in respiration. Too much salt, on the other hand, prevents plant cells from taking up water, drying them out and turning them into shrivelled husks.

To find out how Galdieria survives such extreme environments Schoenknecht and fellow scientists from Oklahoma and the Heinrich-Heine University in Germany decoded the alga's genes. What they found surprised them. Rather than inheriting its superpowers from its ancestors, the algae stole them from bacteria.

This gene-swapping phenomenon is known as "horizontal gene transfer". Usually the genes a lifeform carries are ones it inherited from its parents. This is certainly the case in humans &ndash you can trace back your characteristics along the branches of your family tree to the very first humans.

Schoenknecht identified 75 genes in the algae that were taken from bacteria or archaea

However, it turns out that every now and then "alien" genes from a totally different species can incorporate themselves into DNA. The process is very common in bacteria. Some argue it has even happened in humans, although this has been hotly disputed.

When the foreign DNA lands in its new host, it does not necessarily sit there idly. Instead it can set to work hijacking the host's biology, encouraging it to make new proteins. This can give the host species new skills and allow it to survive in new situations. If the gene jumping happens frequently enough, it can send the host organism down a completely new evolutionary path.

In total Schoenknecht identified 75 genes in the algae that were taken from bacteria or archaea. Not all the genes give the algae an obvious evolutionary advantage, and the exact function of many of the genes is unknown.

However, many of the genes do help Galdieria survive in its extreme environment.

Its ability to cope with toxic chemicals like mercury and arsenic is due to genes taken from bacteria.

One of these genes is for an "arsenic pump" which allows the algae to effectively remove arsenic from its cells. Other stolen genes found include those for metal transporters that allow Galdieria to excrete toxic metals, whilst simultaneously taking up essential metals from its environment. Yet other of the purloined genes control enzymes that allow Galdieria to detoxify metals such as mercury.

The algae had also nicked genes that allow it to tolerate high salt levels. Under normal circumstances, a very salty environment will suck the water out of a cell and kill it. But by synthesising compounds inside the cell that equalise the "osmotic pressure", Galdieria escapes that fate.

It is also a mystery &ndash for now &ndash how Galdieria copes with extreme heat

It is thought that Galdieria's ability to tolerate extremely acidic hot springs is because it is impermeable to protons. In other words, it can simply stop acid from entering its cells. It does this by having fewer genes that code for the channels in the cell membrane through which protons normally pass. These channels usually let in positively charged particles like potassium, which are essential to cells &ndash but they can also let in protons.

"It seems that adaptation to the low pH was mainly driven by removing any membrane transport protein from the plasma membrane that would allow protons to enter the cell," says Schoenknecht. "Most eukaryotes have numerous potassium channels in their plasma membrane, Galdieria has only a single gene encoding a potassium channel. So making the plasma membrane 'proton tight' seems to be the main approach to deal with low pH."

However those potassium channels perform important jobs, such as potassium uptake, or maintaining a voltage difference between the cell and outside. How Galdieria stays healthy without the potassium channels is currently entirely unclear.

It is also a mystery &ndash for now &ndash how Galdieria copes with extreme heat. The scientists were unable to identify genes that could explain that particular feature of its biology.

Bacteria and archaea that can live at extremely high temperatures have distinctly different-looking protein and membranes, but the changes that Galdieria has are probably more subtle, says Schoenknecht. "We have some indications that there are changes in membrane lipid metabolism at different growth temperatures, but we do not yet understand what actually happens, and how it relates to adaptation to heat."

It is clear that copying genes has given Galdieria a huge evolutionary advantage. Whilst most of the unicellular red algae related to G. sulphuraria live in volcanic areas and are somewhat heat- and acid-tolerant, few of its relatives can take as much heat, as much acid, as much toxicity, and as much salt as G. sulphuraria can. In fact, in some places the species makes up 80 to 90% of life &ndash a sign of how challenging other species find the conditions G. sulphuraria calls home.

As soon as a resistant gene occurs, it is quickly swapped between different bacteria

There is one obvious question that remains to be answered: just how has Galdieria managed to steal so many genes?

The algae live in an environment that contains lots of bacteria and archaea so in one sense the opportunity to steal genes is there. However, scientists do not know exactly how the DNA has jumped from bacteria to such a different organism. To successfully get into its host, the DNA must first get into the cell, and then into the nucleus &ndash and then it has to splice itself into the host's genome.

"The current best guess is that viruses might have shuttled the genetic material from bacteria and archaea into Galdieria. But this is a speculation, lacking evidence," says Schoenknecht. "Actually getting into the cell might be the hardest step. Once inside the cell getting into the nucleus and getting integrated into the nuclear genome is probably less of a hurdle."

Horizontal gene transfer happens a lot in bacteria. It is why we have such problems with antibiotic resistance, says Schoenknecht. "As soon as a resistant gene occurs, it is quickly swapped between different bacteria."

However, gene swapping was thought to occur far less frequently in more advanced organisms like eukaryotes. It is thought that bacteria have dedicated uptake systems that allow them to take in nucleic acids, and that eukaryotes lack these.

Nevertheless, other examples of advanced creatures stealing genes to survive in extreme places have been found. A species of snow algae, Chloromonas brevispina, which lives in the snow and ice of Antarctica, carries genes that have likely been taken from bacteria, archaea or even fungi.

Jagged crystals of ice can puncture and perforate cell membranes, so creatures living in cold climates must find some way of dealing with this. One way of doing so is to produce ice-binding proteins (IBP) which when secreted from the cell cling to ice, stopping ice crystals from growing.

The beautiful monarch butterfly may even have nicked genes too

James Raymond from the University of Nevada, Las Vegas, mapped the genome of the snow alga and found that the genes for ice-binding proteins were remarkably similar to those seen in bacteria, archaea and fungi, suggesting that they acquired the ability to survive in cold climates from horizontal gene transfer.

"The genes appear to be essential for survival because they have been found in every ice-associated alga examined so far and not in any algae from warmer habitats," says Raymond.

There are a few other examples of horizontal gene transfer in eukaryotes.

Tiny crustaceans that live in Antarctic sea ice seem to have acquired the skill. The critters &ndashStephos longipes &ndash are able to survive in liquid brine channels in the ice.

"In field measurements I found S. longipes living in supercooled brines within the surface layer of sea ice," says Rainer Kiko, a scientist at the Institute for Polar Ecology at the University of Kiel, Germany. "Supercooled means that the temperature of the liquid is below the freezing point you would expect it to be based on how salty it is."

To survive and stop itself from freezing, S. longipes's blood and all other body liquids contain the same amount of molecules as the surrounding medium, which lowers its freezing point to match that of the water around it. However, the crustacean also produces antifreeze proteins that prevent ice crystals from forming in its blood.

The sea slug can survive using the energy from sunlight

The proteins, which are not found in related crustaceans, are very similar to those found in sea ice algae, suggesting this protein was obtained through horizontal gene transfer.

The braconid wasp is famous for injecting an egg along with a virus into a host insect. The viral DNA hijacks the host's brain, turning the host into a zombie, which then acts as an incubator for the wasp egg. Scientists found braconid genes in butterflies, even when they had never been colonised by wasps. The genes are thought to make butterflies more resistant to disease.

It is not just individual genes that eukaryotes have stolen. Sometimes they go in for theft on a grander scale.

A bright green sea slug called Elysia chlorotica is thought to have acquired the ability to photosynthesise through eating algae. The sea slugs swallow chloroplasts &ndash organelles that perform photosynthesis &ndash whole and keep them stored in digestive glands. Then if the going gets tough and there are no more algae to eat, the sea slug can survive using the energy from sunlight to convert carbon dioxide and water into food.

One study suggests the sea slugs have also directly taken genes from the algae. Scientists inserted fluorescent DNA markers into the algal genome, so they could see exactly where the genes were. After feeding on the algae, the sea slug acquired a gene responsible for repairing chloroplasts.

It may be that stealing genes is a fairly common evolutionary tactic

Meanwhile, the cells in our bodies contain tiny energy-generating structures called mitochondria that look distinct from the rest of our cellular structures. Mitochondria even have their own DNA.

A leading theory is that mitochondria existed as independent lifeforms billions of years ago, and that they somehow became incorporated into the cells of the first eukaryotes &ndash perhaps the mitochondria were swallowed but somehow escaped being digested. This event is thought to have happened about 1.5 billion years ago, and was a key milestone in the evolution of all higher life forms such as plants and animals.

It may be that stealing genes is a fairly common evolutionary tactic. After all, it makes sense to let others do all the hard work for you whilst you nip in at the end and reap the rewards. Alternatively, horizontal gene transfer may speed up an evolutionary journey already in progress.

"An organism that is not adapted to heat or acid at all will probably not suddenly inhabit volcanic pools because it acquired the right genes," says Schoenknecht.

"However, evolution is almost always a stepwise process, and with horizontal gene transfer these steps forward might be a bit larger."

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Neanderthals possessed crucial gene linked to speech, DNA evidence suggests

NEW YORK — Neanderthals, an archaic human species that dominated Europe until the arrival of modern humans about 45,000 years ago, possessed a critical gene known to underlie speech, according to DNA evidence retrieved from two individuals excavated from El Sidron, a cave in northern Spain.

The evidence stems from analysis of a gene called FOXP2 which is associated with language. The human version of the gene differs at two critical points from the chimpanzee version, suggesting that these two changes have something to do with the fact that people can speak and chimps cannot.

The genes of Neanderthals seemed to have passed into oblivion when they vanished from their last refuges in Spain and Portugal 30,000 years ago, almost certainly driven to extinction by modern humans. But recent work by Svante Paabo, a biologist at the Max Planck Institute for Evolutionary Anthropology in Leipzig, Germany, it has made clear that some Neanderthal DNA can be extracted from fossils.

Paabo, Johannes Krause, an anthropologist, and Spanish colleagues who excavated the new bones say they have now extracted the Neanderthal version of the relevant part of the FOXP2 gene. It is the same as the human version, they report in the Friday issue of Current Biology.

Because many other genes are also involved in the faculty of speech, new finding suggests but does not prove that Neanderthals had human-like language.

"There is no reason to think Neanderthals couldn't speak like humans with respect to FOXP2, but obviously there are many other genes involved in language and speech," Paabo said.

The human version of the FOXP2 gene apparently swept through the human population before the Neanderthal and modern human lineages split apart 350,000 years ago.

But until more is known about what FOXP2 does in the brain, it is hard to know what powers were conferred by the sweep, said Gary Marcus, a psychologist at New York University who has written about the evolution of language. "Perhaps Neanderthals had some rudiments of language, but then again, maybe not."

A new strain of mice may have something to say about how FOXP2 affects language. Paabo has developed mice whose FOXP2 genes have been replaced with the human version. The mice have extra neuronal connections in their brains and make an unusual sound. "There seems to be a change in vocalization - they squeak in a different way," Paabo said. "But there are no obvious differences in behavior - in most ways they are normal mice."

The ability to fish out a specific gene of interest from the Neanderthal genome is a remarkable technical feat, if that has indeed been achieved. The results "have the potential to become a keystone in our understanding of human evolution," wrote an anonymous referee who reviewed Paabo's report for Current Biology.

The study of human evolution may take a giant leap forward if Paabo should recover the entire Neanderthal genome, at least in draft form, a feat he said he hoped to accomplish by next year.

But two sudden clouds have overshadowed this grand prospect. One is that the new finding about FOXP2 sharply contradicts an earlier result that Paabo announced five years ago.

Surveying the human version of FOXP2 in populations around the world, Paabo found in 2002 that everyone had essentially the same version of the gene. This happens when a new version of a gene confers such a survival advantage that it sweeps through the population. This sweep had occurred sometime within the last 200,000 years, Paabo and colleagues reported in an article in Nature.

That date supported a proposal by Richard Klein of Stanford University, based on archaeological evidence, that the modern human population had undergone some neurological change around 50,000 years ago, which enabled their populations to expand and emerge from Africa. The neurological change could have been the perfection of modern language, given that few evolutionary advances could be more valuable to a social species.

But Paabo's new report pushes back the language-related changes in FOXP2 to at least 350,000 years ago, the time that the Neanderthal and modern human lineages split, a date that no longer supports Klein's thesis.

Pushed by the referees of his new report to say why the old date was so wrong, Paabo told the editors of Current Biology that the calculations underlying the younger date were "not flawed but rely on assumptions that are necessary but also universally known to be oversimplifications of the reality."

While the assumptions may be well known to population geneticists, the caveats were not so clear to others. Klein said he was disappointed to have lost the genetic support from Paabo's work but had not changed his views. "The archaeological record suggests a major change in human behavior 50,000 years ago," he said, "and I think there is overwhelming evidence for that."

A second cloud over Paabo's work with Neanderthal DNA is the ever-present danger of contamination with the human DNA, especially since Paabo reports finding the human version of FOXP2 in Neanderthal bones.

Most fossil bones in museum collections, and even the chemical reagents used to analyze genetic material, are contaminated with human DNA. The contaminant often overwhelms the faint residual traces of Neanderthal DNA, which is hard at best to tell apart since the sequence of units is so similar.

Paabo has struggled valiantly to cope with the contamination issue. He has recovered the DNA sequence of Neanderthal mitochondrial DNA, a kind that is separate from the main genome in the cell's nucleus. By measuring the ratio of Neanderthal to human mitochondrial DNA, he can assess the degree of contamination in a sample.

Last year, to lay the groundwork for his analysis of the entire Neanderthal genome, Paabo decoded the sequence of many DNA fragments, and sent samples to a second laboratory for independent analysis.

This seemed a considerable feat. But in an article soon to be published in the journal PLoS Genetics, Jeffrey D. Wall and Sung K. Kim, two biologists at the University of California, San Francisco, say there are serious inconsistencies between the Neanderthal sequences that Paabo published last year and those of the second laboratory, the Joint Genome Center Institute in Walnut Creek, California, headed by Edward M. Rubin.

The bottom line of their analysis is that Rubin's results were probably correct and that Paabo's were highly contaminated with human DNA.

Paabo said he agreed in general with Wall and Kim's criticisms but noted the DNA extracts for both studies had been made in his clean room. He had then sent the samples for his own analysis to another laboratory, where the contamination could perhaps have occurred.

Paabo has now added extra safeguards, he said, such as tagging all the Neanderthal DNA extracted in his clean room.

For the FOXP2 analysis, he and his Spanish colleagues arranged for the bones to be excavated under sterile conditions and immediately frozen. In addition he analyzed the Neanderthal Y chromosome, showing it was very different from the human Y chromosome, and so provided a second test along with mitochondrial DNA to differentiate human and Neanderthal samples.

Wall said that in the new report Paabo and his colleagues "have been much more careful than they were before to control contamination, but I think it still remains a small possibility."

Why was such a striking result not presented to a better known journal such as Nature? Paabo replied that he had done so, but that "Nature rejected it without review. I was surprised."

FOXP2 first came to light in a large London family, half of whose members had subtle defects in their speech and understanding. Geneticists discovered that one of their two copies of FOXP2 was inactivated by a mutation.

The gene "provides an exciting molecular window into brain circuits that are important in speech," said Simon Fisher of Oxford University, a member of the team that discovered the FOXP2 mutation. Neanderthals and mice are not the only species contributing to the discussion. Echo-locating bats have a distinctive change in their FOXP2 gene at the same location as the human changes. Bats that don't hunt with sonar do not have these changes, a team of Chinese biologists, led by Gang Li and Shuyi Zhang of the East China Normal University in Shanghai, report in the current issue of the journal PLoS One.

This suggests FOXP2 may have evolved in bats to support the rapid motor sequencing involved in echolocation. Similar tweaking of FOXP2 could have occurred in the human lineage to support the fine motor sequencing involved in speech, Fisher said.

Is Biologist Barry Commoner a Mutant?

Longtime radical activist and biologist Barry Commoner has just published a preposterous article in the February issue of Harper's that purports to call into question the "foundation of genetic engineering." Commoner claims to have uncovered a scandal at the heart of biology and biotechnology that the leading researchers and greedy corporations are keeping hidden from an unwitting public.

What is this dark secret? That a gene can specify more than one protein through alternative splicing. According to Commoner, alternative splicing contradicts what biologists often call the Central Dogma in which a DNA gene is transcribed into messenger RNA (mRNA) that is then translated into a string of amino acids that folds to form proteins. Proteins are the molecular machines at the heart of most processes in living cells.

The Human Genome Project's findings highlighted the importance of alternative splicing, according to Commoner. Recall that last year two teams, one private led by Craig Venter and the other government-run led by Francis Collins, jointly announced that they had decoded the human genome, the 3.2 billion DNA base pair recipe for making human beings. Preliminary analyses indicate that the recipe for making a human being is composed of 30,000 to 40,000 genes. But it is estimated that there are at least 100,000 different proteins in human bodies, so that means that some genes must be recipes for more than one protein.

To understand how this happens, please bear with me for a short lesson in molecular biology. Genes are not neat orderly sequences of DNA bases that are simply read off one by one. Instead, the DNA bases that make up a gene–called exons–are often interrupted by other DNA bases called introns that have nothing to do with the gene. In the first step in transcribing DNA into RNA, both exons and introns are read off to produce pre-messenger RNA. To get the proper recipe for a protein, the introns must be removed. That feat is accomplished by an editing machine composed of RNA and protein called the spliceosome that removes the introns and splices together the exons into mature messenger RNA that now embodies the proper recipe for a specific protein.

Alternative splicing occurs when regulatory elements in the genome perhaps tell the spliceosome to treat some introns as exons or some exons as introns, thus changing the protein recipe. As University of Georgia biologist Wayne Parrott notes, to a certain extent this is all a matter of nomenclature—is it the "same" gene that is specifying different proteins or are they really different genes that happen to share overlapping DNA sequences? The fact is "there is still one DNA sequence per protein," says Parrott.

In this quibble over nomenclature, Barry Commoner sees far darker implications. "The discovery of alternative splicing…bluntly contradicts the precept that motivated the genome project," writes Commoner. "It nullifies the exclusiveness of the gene's hold on the molecular process of inheritance and disproves that by counting genes one can specify the array of proteins that define the scope of human inheritance. The gene's effect on inheritance thus cannot be predicted simply from its nucleotide sequence…."

First, the genome project is not just about "counting" genes. Second, researchers must find all the genes in order to have any idea what protein(s) they might express, alternatively spliced or not. The human genome (the set of all genes) is the gateway to the human proteome (the set of all proteins). Commoner wants to claim that researchers have ignored what he believes is the crucial role that protein and RNA molecular "machines" play in expressing inheritable traits.

But that is sheer nonsense as a quick glance at any freshman-level biology textbook shows. For example, Molecular Cell Biology 4th Edition by Lodish et alia, states: "A more accurate way of representing the relationship between the synthesis of DNA, RNA, and proteins in all cells would look like [the figure below], indicating that special proteins catalyze the synthesis of both RNA and DNA."

Next, Commoner asserts that alternative splicing "destroys the theoretical foundation of a multibillion-dollar industry, the genetic engineering of food crops." Because of alternative splicing, genetically enhanced crops "represent a massive uncontrolled experiment whose outcome is inherently unpredictable. The results could be catastrophic." Commoner is canny enough to know that his unusual views on protein synthesis and alternative splicing would be simply ignored by modern researchers unless he can garner attention for them. So he links his views to the activist campaign against plant biotechnology and publishes them in a popular magazine. (One may be forgiven for thinking that Commoner's article appeared in Harper's because no reputable peer-reviewed scientific journal would be likely to publish it.)

Does alternative splicing really destroy the biotech industry and threaten human health?

Commoner begins his article by citing problems with gene-transfer experiments in animals and with cloning (in which no genes are transferred). He then blithely notes in the same paragraph that some non-plant genes have been transferred into commercial crops. Commoner clearly hopes that readers will make the leap of assuming that if there are problems in animal biotech experiments then there must also be problems in commercial plant biotech. He has to use this sneaky rhetorical technique because there simply aren't any "failures" in commercial plant biotechnology he can cite.

Commoner's chief claim is that adding genes for traits such as bacterial pesticides to crops such as corn and soybeans is dangerous because in an "alien genetic environment, alternative splicing of the bacterial gene might give rise to multiple variants of the intended protein—or even to proteins bearing little structural relationship to the original one, with unpredictable effects on ecosystems and human health." He argues that the plant protein complexes that guide RNA and protein synthesis will not be able to properly transcribe and translate bacterial genes, so unintended proteins will be produced.

Has Commoner any evidence that this occurs frequently or at all in commercial biotech crops? If he does, he doesn't cite it in Harper's. In fact, plant biotechnologists are quite adept at adapting bacterial genes to plant genomes so that their protein synthesis machinery functions normally. If it didn't function normally, the plants wouldn't produce the traits such as pest resistance that commercial biotech companies are so successfully selling to farmers around the world. To produce a commercial biotech crop variety, biotechnologists typically begin by producing hundreds and thousands of plants in which they are trying to insert a particular gene. Over the years they grow and select the ones in which the trait they are seeking—say, pest resistance–is stable. Only after years of testing and research will they commercialize the selected crop variety.

Commoner also claims, "The degree to which disruptions (caused by gene transfers) do occur in genetically modified crops is not known at present, because the biotechnology industry is not required to provide even the most basic information about the actual composition of the transgenic plants to the regulatory agencies."

"Outright false," says biologist Parrott. "Transgenics cannot be different from conventional varieties." Biotech crops must be "substantially equivalent" to conventional varieties before they can be marketed. In every case, biotech companies have submitted reams of information to the Food and Drug Administration (FDA) on things like nutrient profiles and feeding values before marketing genetically enhanced crops.

Conventional plant breeders don't need FDA approval to market their crops, even though they often involve far more genetic changes (through techniques like crossbreeding) than the single-gene transfers common in genetically enhanced crops. "Every single differently shaped leaf of lettuce, every different color of bell pepper, every new variety of citrus fruit, is the result of genetic mutations that produce different proteins which were noticed and then selected by conventional plant breeders," says Parrott. Yet no one worries about being poisoned by these far more massive genetic alterations in crops. Interestingly, scores of varieties of crops being grown today were produced through mutations induced by radiation and caustic chemicals in the 1940s and 1950s. No one knows what proteins these random genetic mutations produced, but people have been eating them for half a century without ill effects.

Parrott points out that plant genomes are filled with DNA fragments called retrotransposons that naturally jump randomly from one part of a plant's genome to another. These jumps occur billions of times every growing season. They often disrupt gene expression in plants and may well sometimes induce the production of novel proteins. But this is no cause for alarm, since people have been eating these crops with their jumping genomes for centuries. It is evident that such disruptions in plant genomes have an extremely low probability of producing any dangerous proteins.

Why is Commoner making claims that can be refuted simply by referring to college biology textbooks? Partly because he is still smarting from his intellectual defeat at the hands of James Watson and Francis Crick (the co-discoverers of the structure of DNA) in the 1950s and 1960s. At that time, Commoner, besides being a campaigner against aboveground nuclear testing, was one of the leading advocates of the theory that proteins carried inheritable traits. To some extent, Commoner appears to be trying to reinterpret alternative splicing as a way to rescue at least a portion of his old thesis. It turns out that Commoner may be a perfect example of Thomas Kuhn's contention in The Structure of Scientific Revolutions that old theories never die until old theorists do.

Bangladesh decodes Jute Plant Genome

Bangladeshi researchers have successfully decoded the Jute Plant Genome.

With the successful sequencing of jute genome , Bangladesh has become only the second country after Malaysia, among the developing nations, to achieve such a feat.

Researchers from Dhaka University, Bangladesh Jute Research Institute and Software Company DataSoft in collaboration with Centre for Chemical Biology University of Science Malaysia and University of Hawaii, USA has decoded the genome.

Prime Minister Sheikh Hasina made the announcement of Bangladesh&#8217s scientific adherence in the Parliament on Wednesday.

Dubbing it a &#8216historic scientific advancement&#8217, Sheikh Hasina said the discovery would rejuvenate the lost heritage of &#8216golden fibre&#8217 as gene mapping of jute would now help breeders develop jute varieties resistant to pests and climate adversities



P lant Tissue Cult. & Biotech. 15(2): 145-156, 2005 (December)

Preliminary Progress in Jute (Corchorus species)
Genome Analysis

Ahmad S. Islam, Matthew Taliaferro, Christopher T. Lee, Craig
Ingram, Rebecca J. Montalvo, Gerrit van der Ende, Shahabudin
Alam1, Javed Siddiqui1
and Kanagasabapathi Sathasivan*

Molecular Cell and Developmental
Biology, School of Biological Sciences, The University
of Texas at Austin, USA

Key words: Genomic DNA, Corchorus olitorius, C. capsularis, WU-BLAST, TAIR,
18S rRNA, tRNA-Leu

The paper summarizes the progress made in cloning and sequencing a limited
number of genes from jute (Corchorus olitorius and C. capsularis) and discusses
future applications of jute genome analysis. As of December 2005, slightly over
200 DNA sequences have been deposited in GenBank. Although many of these
sequences are partial and uncharacterized, this marks the beginning of a major
step in unraveling of the hitherto unknown jute genome. We have constructed
both cDNA and genomic DNA libraries for C. olitorius var. O4 and C. capsularis
var. CVL-1 in the plasmid vectors pSMART and pBluescript, respectively.
Random clones were isolated and sequenced. These DNA sequences have been
deposited in GenBank and analyzed using TAIR (TAIR - Home Page) for
similar sequences in Arabidopsis thaliana and other related plant species. The
complete sequence for tRNA-Leu and partial sequence of DNA fragments
encoding several proteins, such as RNA polymerase &#946 subunit-1, 18S rRNA,
mitochondrial DNA directed RNA polymerase and carboxytransferase &#946-subunit
are reported in this paper. The analysis of DNA sequences of related taxa
deposited in GenBank is also presented delineating the scope and applications of
cloning genes of agronomic importance.

Jute, the world&#8217s second most cultivated fiber crop next to cotton, is extensively
grown in India, Bangladesh, China, Thailand, Russia, Myanmar, Nepal,
Uzbekistan, Chile and Brazil (FAOSTAT). India and Bangladesh
rank as the top two countries in terms of jute production. In Bangladesh, jute is
the principal cash crop and jute products contribute a significant portion to

*Corresponding author ([email protected]) 1DNA Technologies, Gaithersburg, Maryland. 146 Islam et al.
country&#8217s foreign exchange ( Jute
cultivation helps sustain millions of farmers in these two countries alone. The
cultivated varieties of jute have been evolved from Corchorus olitorius L. and C.
capsularis L. through conventional breeding and pure line selection (Ghosh 1983)
based on their yield and agronomic performance. Synthetic fibers have been a
major competition in the international market to the natural jute fibers. In recent
years the situation has improved because natural fibers do not pollute the
atmosphere as do synthetics and may help reduce the cutting of trees for making
paper. In addition, jute is now being utilized to manufacture more value-added
industrial products such as in the making of Geo-textiles (Dedicated Servers | Managed Dedicated Servers | Web Hosting | VPS | Liquid Web. for protecting embankments against river erosion, fiber
reinforced building materials, packaging materials and in the production of
paper. However, there are problems in increasing the productivity and
profitability of jute. Some of the major challenges include susceptibility of the
jute crop to insect pests and fungal diseases, photoperiod sensitivity, poor fiber
quality, and low yield under unfavorable growth conditions such as salinity,
drought, flood or cold. Addressing these challenges through traditional plant
breeding program has limitations due to lack of genetic diversity among
cultivated jute varieties and sexual incompatibilities between the cultivars of the
two jute species and between each of the two species and wild Corchorus species.

In spite of developing successful hybrids between two species of jute namely, C.
olitorius and C. capsularis (Islam and Rashid 1960), it was not possible to release
any variety from the advanced progeny of the above interspecific crosses.
Hence, in recent years molecular approaches to improve the agronomic traits of
jute are being considered as alternatives.

Recently, research in some universities in the Indian subcontinent has
resorted to molecular approaches through systematic cloning and transgenic
work. By means of a combined study of AFLP and RAPD, Hossain et al. (2002,
2003) at Dhaka University have shown the importance of using molecular
markers in distinguishing between cold-tolerant and cold sensitive jute varieties
obtained from GenBank at Bangladesh Jute Research Institute (BJRI). Using
simple sequence repeat (SSR) marker loci and AFLP assay, Basu et al. (2004) at
the Indian Institute of Technology (IIT), Kharagpur, evaluated genetic diversity
of 49 genotypes of the two jute species. More recently, P. K. Gupta at Charan
Singh University, Meerat (CSUM), India and his associates as well as the Dhaka
University team led by Haseena Khan developed genomic SSRs and deposited
the sequences in GenBank. By developing more SSRs Gupta and his team at
CSUM are planning to embark upon a program of gene tagging combined with
the construction of a framework linkage map for QTL interval mapping. In
addition to the research on markers, procedure for successful regeneration (Seraj Preliminary Progress in Jute (Corchorus spp.) Genome Analysis 147
et al. 1992 Saha et al. 1999) and transformation of C. capsularis (Ghosh et al. 2002)
have been developed.

At the University of Texas at Austin, work on the construction of cDNA and
genomic DNA library of the above two jute species has been initiated recently.
As a first step towards achieving this goal, a rapid method for high quality RNA
isolation from jute was developed in this lab (Khan et al. 2004). A cDNA and
genomic DNA libraries have been constructed in pSMART and pBluescript
vectors from C. olitorius and C. capsularis, respectively. We have isolated and
sequenced random fragments with the objective of testing these DNA libraries
and deposited some of the sequences in GenBank. This paper reports analysis of
the DNA sequences that show homology with other plant genes including that of
Arabidopsis thaliana. It summarizes the results from the analysis of selected DNA
sequences currently available in GenBank. We present the challenges
encountered in cloning genes from jute and suggest possible solutions to
overcome such problems. In addition, this paper attempts to update jute
researchers on the current status in jute molecular biology and to establish liaison
with any other team(s) who may be interested to collaborate in the jute genome

Material and Methods
Plant materials and growth: Seeds of C. capsularis var. CVL-1 and of C. olitorius var.
O4 were supplied by Bangladesh Jute Research Institute through the courtesy of
Haseena Khan, Dhaka University. Seeds from C. olitorius were surface sterilized
with 10 &#37 bleach and incubated on a moist filter paper in Petri dishes inside an
incubator at room temperature (23&#176C). Seven-day-old seedlings grown inside
Petri dishes were collected and shipped to DNA technologies, Maryland for
RNA isolation and cDNA library construction. The sample weight was roughly
1g. Seeds of C. capsularis were surface sterilized in the same manner and planted
in the greenhouse under normal lighting and allowed to grow to mature plants.
The young leaves of mature plants were harvested, frozen in liquid nitrogen and
shipped to DNA technologies, Maryland for genomic DNA isolation and library

Isolation of the total RNA: The frozen jute tissues were directly grinded in the
RNA isolation solution (Trizol-Invitrogen) in liquid nitrogen. The lysates were
centrifuged at 10,000 &#215 g for 10 min to remove unlysed cells and contaminants.
The supernatant was mixed with 0.2 volume of chloroform and incubated on ice
for 15 min. The mixture was centrifuged at 10,000 &#215 g for 10 min and the upper
layer was collected. The upper layer was mixed with half volume of isopropanol
and centrifuged for 30 min at 10,000 &#215 g. The pellet was washed with 70% ethanol 148 Islam et al.
and air dried. The RNA was dissolved in DEPC treated autoclaved water. An
aliquot of RNA was checked on denaturing agarose gel.
Isolation of the mRNA: Jute mRNA was isolated from total RNA using oligo
dT cellulose. Briefly, the total RNA was mixed with 10 ml of binding buffer (10
mM Tris pH 7.5, 500 mM NaCl) and heated at 70&#176C for 5 min. The samples were
immediately chilled on ice for 5 min and mixed with oligo dT latex beads. The
mixture was incubated at room temperature for 2 h to allow the complete
binding of the mRNA with oligo dT. After incubation, the mixture was
centrifuged at 5,000 &#215g. The pellet was washed twice with the binding buffer.
Now, the pellet was washed with excess of a low salt buffer (10 mM Tris pH 7.5,
250 mM NaCl). This step was repeated several times to remove all unbound
RNA molecules. The mRNA was finally eluted with 10 mM Tris pH 8.0. An
aliquot of mRNA sample was checked on denaturing agarose gel.

Construction of the cDNA library: Two &#181g (micrograms) of mRNA were mixed
with oligo dT primer (18 mer) and heated at 65&#176C for 10 min. The mixture was
cooled on ice and the following cDNA synthesis reagents were added: dNTP,
RNase inhibitor, first strand cDNA synthesis buffer and superscript reverse
transcriptase. The reaction mixture was incubated at 42&#176C for 2.5 h. The second
strands were synthesized using dNTP mix, E. coli DNA polymerase and RNase H
in second strand buffer at 16&#176C for 2h. After the second strand synthesis, the
ends were polished using Pfu polymerase at 72&#176C for 30 min. The cDNA was
extracted with phenol: chloroform: isoamyl alcohol.

EcoRI adapter ligation, kinasing of cDNA ends and size fractionation of the cDNA:
The end-polished cDNA was mixed with EcoRI adapter, T4 DNA ligase buffer
and a high concentration of T4 DNA ligase. The mixture was incubated at 8&#176C
for 48h. The mixture was heated at 50o
C for 5 min. The ends of cDNA were
phosphorylated (addition of 5&#8217-phosphate) by T4 kinase. The reaction was
continued at 37&#176 C for 1 h. The kinased cDNA was precipitated with ethanol and
3M sodium acetate. The adapter ligated cDNA was re-suspended in 10 mM TE
buffer and electrophoresed on 1% low melting point agarose gel for 3 h. The
cDNA in the size range of 0.5 kb and higher was recovered from low melting
point agarose by extraction with phenol phenol chloroform and chloroform. The
cDNA was precipitated with ethanol.

Vector digestion, purification and the ligation of cDNA: For making the library,
pSMART vector from Lucigen was used. The sequences and the restriction map
of the vector are available in Advanced Products for Molecular Biology - Lucigen Corporation. The vector was digested with
EcoRI and treated with calf intestinal alkaline phosphatase (CIAP). The digested
vector was purified on the agarose gel. An aliquot of digested vector was ligated
and used to electroporate E. coli DH10B cells to test the efficiency of the
digestion. After getting successful results, the cDNA was separately ligated into Preliminary Progress in Jute (Corchorus spp.) Genome Analysis 149
the vector in the presence of T4 DNA ligase at various concentrations. The
samples were incubated at 8&#176C for 48 h. Ligated cDNA was precipitated with
ethanol and re-suspended in water. An aliquot of the ligated samples were
electroporated in E. coli DH10B cells. Immediately after electroporation, the cells
were mixed with SOC medium and allowed to recover for 1.5 h at 37&#176C. Libraries
were centrifuged at 5000 rpm for 10 min. The medium was discarded and the
pellet was re-suspended in SOC medium containing the appropriate antibiotic
and 15% glycerol. The cells were plated on LB ampicillin plates and grown at
37&#176C overnight. DNA from single colonies was isolated and screened for the
presence of inserts.

Genomic DNA library construction: Pre-chilled (&#8211 80&#176C) sterile grinder and
pestle were used to grind young jute (C. olitorius var. O-4) leaves until the leaf
became powdery. Five ml of lysis buffer-I containing proteinase K were added to
the powdered leaves and the mixture was incubated at 37&#176C in a shaker
overnight with a low-speed agitation. The incubated material was spun at 10,000
rpm for 15 min at 4&#176C and the supernatant was collected. After adding 5 ml
phenol : chloroform : isoamyl alcohol (PCI) to the supernatant, it was vortexed
and spun at 10,000 rpm for 5 min. The aqueous phase was saved and 3M sodium
acetate (0.3M final) was added to it. Nearly 2.5 volumes of 100% ethanol (cold)
were added to the aqueous phase the material was well mixed and stored at
&#821120&#176C for 30 min. The re-suspended pellet was spun down after 1 ml of TE was
added to it. Genomic DNA was then isolated by Easy-DNA Isolation Kit
(Invitrogen, Carlsbad, CA (cf. manufacturer&#8217s protocol). In short, 350 &#181l of
solution A were added to the DNA and incubated at 65&#176C for 10 min. Thereafter,
150 &#181l of solution B were added and the mixture was vortexed vigorously until
the precipitation was found to move freely. After adding 500 &#181l chloroform to
the previous mixture, it was vortexed until the viscosity of the material was
found to decrease. The next step was centrifugation at 14,000 rpm for 20 min. To
the upper transferred aqueous phase, 1 ml of cold 100% ethanol was added,
mixed well and left at &#8211 20&#176C for 30 min. The centrifugation at 14,000 rpm for 20
min was repeated. To the transferred upper aqueous phase, 500 &#181l of cold 80%
ethanol were added and spun at 14,000 rpm for 5 min. The pellet was re-
suspended in 100 &#181l TE containing RNase. The last step was incubation at 37&#176C
for 30 min and the incubated product was stored at 4&#176C. The quality of the
isolated genomic DNA (gDNA) was checked by running the samples on a 0.7%
agarose gel and taking OD readings at 260 nm.

Restriction enzyme digestion of gDNA and size fractionation: Genomic DNA was
partially digested with restriction enzyme Apo I and Sma I (Roche Diagnostics,
Indianapolis, IN) in separate tubes for ligation with their specific vectors.
Approximately, an amount of 10 &#181g of gDNA was taken for restriction enzyme 150 Islam et al.
digestion for 90 min about one-third of the partially digested gDNA was
removed from the digestion reaction and mixed with 10 &#181l of 0.5 M EDTA every
30 min, followed by their storage at 4&#176C. The three restriction enzyme-digested
DNA fractions were combined. The next step was to purify the digested material
using Qiagen MinElute Gel extraction Kit (cf. manufacturer&#8217s protocol).

Vector preparation: Seven &#181g of pBluescript SK (+/-) vector (www.Stratagene.
com) was digested twice with EcoRI and SmaI (Roche Diagnostics, Indianapolis,
IN) in separate tubes for the ligation with their specific inserts. Briefly, the DNA
was digested for 2.5 h at the specific temperature thereafter more enzymes were
added and the digestion period was prolonged for another 2 h. The digested
DNA was inactivated by heating at 70&#176C for 10 min and treated with Shrimp
alkaline phosphatase, (SAP Roche Diagnostics, Indianapolis, IN) after it had
cooled down to room temperature, (cf. the manufacturer&#8217s protocol). Restriction-
enzyme digested and SAP-treated vector DNA was then purified with PCI
followed by treatment with chloroform: isoamyl alcohol (CIA), and finally
precipitated with ethanol. The DNA was then re-digested with the same set of
enzymes and the same time frame as above followed by SAP treatment, purified
with PCI followed by CIA and precipitated with ethanol. The vector DNA was
re-suspended with an appropriate amount of sterile H2O/TE. The quality check
(QC) of the isolated vectors was performed by running the samples on a 0.7%
agarose gel.

Ligation and electroporation: The ApoI digested gDNA was ligated with the
pBluescript SK (+/-) vector which was earlier digested with EcoRI and the Sma I
digested gDNA was ligated with the pBluescript SK (+/-) vector which was
earlier digested with Sma I, respectively. The ligation reaction was made for 10 &#181l
and incubated for two days at 4&#176C. Two &#181l of the ligated reaction mixture were
added to 70 &#181l of Electro-10 Blue competent cells (thawed at 4&#176C) and
electroporated in a 0.1 cm gap pre-chilled cuvette at 1700 volts. One ml of SOC
was added immediately. Cells were transferred to a new 15 ml culture tube,
incubated at 37oC for 1 h and 100 &#181l of cells were spread on LB-agar plates
containing ampicillin. The plates were incubated at 37&#176C overnight. The number
of colonies was counted.

Quality control (QC) of the library: Ten colonies were randomly picked up and
grown in LB (+ antibiotic) overnight. QC was performed by restriction enzyme
digestion (Fig. 1) or PCR methods. Once the pilot ligations were found effective
(> 90% recombinants), large-scale ligations were set up to get at least 106 primary
clones. The whole library was recovered in 10 ml of SOC at 37&#176C. One hundred
&#181l of the library were plated on LB-plates (with ampicillin). The plates were
incubated overnight. On the following day, the number of colonies was counted.
The library was centrifuged and re-suspended in 2 ml of LB-antibiotic. Approxi- Preliminary Progress in Jute (Corchorus spp.) Genome Analysis 151
mately, 40 - 50,000 colonies were spread on each 150 mm plate. The plates were
incubated overnight. The last step consisted of aliquoting the libraries in 1 ml
aliquot, labeling each container and storing them at &#8211 70&#176C.

Isolation of plasmid DNA: The plasmids containing either cDNA or gDNA
cloned materials were isolated via alkaline lysis procedure through Qiagen spin
columns as per manufacturer&#8217s instructions. Once these plasmids were isolated,
the quantity and quality of the samples were determined based on spectrophoto-
metric (NanoDrop) analysis. In order to identify which plasmids possibly
contained cDNA inserts, we digested each plasmid sample with EcoRV and
EcoRI restriction enzymes and then separated the results of the digest on a 0.8%
agarose gel stained with ethidium bromide, SyBr Green or SyBr Gold. The
recombinant plasmids were identified and sent for sequencing at the core DNA
sequencing facility at The University of Texas at Austin. Upon obtaining positive
sequencing results, a BLAST nucleotide search was performed on each sample in
order to determine to which genes the fragments were most closely related. In
addition, we have confirmed the sequence alignment results with the WU-
BLAST service in TAIR (TAIR - Home Page) and the results have been

Results and Discussion
The cDNA library constructed in pSMART cloning vector (Advanced Products for Molecular Biology - Lucigen Corporation)
into the NotI/Blunt restriction site and stably maintained in the E. coli host cell
DH10B-T1 contained 106 primary clones comprising about 90% recombinants.
The average insert size as determined by PCR was 100 to 500 bp. The inserts
were relatively small ranging from 100 to 500 bp. The short strands of cDNA
could possibly be due to some inhibitory compound that might have co-purified

Fig. 1. Results from gDNA library ten randomly picked clones digested with
Pvu II indicating the presence of DNA inserts in nine out of ten samples.
with the jute RNA during the process of isolation. Special attention to prevent
such contamination may help in the future cDNA synthesis and library
MW 1 2 3 4 5 6 7 8 9 10
3 kbp152 Islam et al.
construction. Random cDNA library clones were cultured on LB kanamycin and
the DNA isolated was sequenced.
The total genomic DNA of C. capsularis was used in the genomic DNA
library constructed in pBluescript KS (Genomics Master). The genomic
library contained 106 to 107 primary clones and the average insert size was about
100 to 1000 bp. Random clones were cultured in LB ampicillin and the plasmid
DNA isolated was sent for sequencing. The cDNA and gDNA sequences were
used to search for similar sequences through the TAIR (TAIR - Home Page)
web site through WU-BLAST (Wisconsin University - BLAST). The sequence
analysis from randomly selected clones is summarized in the Table 1.

Table 1. Examples of the DNA sequences obtained from the jute genomic DNA and
cDNA sequences. Repetitive gene names are mentioned here to illustrate the
frequency of such clones in the library.

TAIR result Score E value
AT1G68990. DNA-directed RNA polymerase,
13 AT3G41768. 18SrRNA 287 2.00E-76
14 AT3G41768. 18SrRNA 344 4.00E-93
5 ATCG00180. RNA polymerase &#946 subunit-1 1068 0
4 ATCG00180. RNA polymerase &#946 subunit-1 337 2.00E-91
9 ATCG00180. RNA polymerase &#946' subunit-1 1207 0
11 ATCG00180. RNA polymerase &#946' subunit-1 371 1.00E-101
15 ATCG00490. large subunit of RUBISCO. 492 1.00E-138
3 ATCG00500. carboxytranserase &#946 subunit 151 3.00E-35
2 ATCG00860. hypothetical protein 101 3.00E-20
8 ATCG00860. hypothetical protein 898 0
ATCG00905. chloroplast gene encoding ribosomal
protein s12
12 ATCG01180. chloroplast-encoded 23S ribosomal RNA 315 9.00E-85
6 ATCG01280. hypothetical protein 870 0
1 tRNA-Leu 161 3.00E-38

The information in the above Table was compiled from all sequences
collected from June to November 2005. All sequences were compared against
known sequences in the TAIR database (TAIR - BLAST) with
introns and untranslated regions included (AGI Genes (+introns. +UTRs)
(DNA)). Any entered sequence that gave homologous sequences with a
maximum score of 50 bits or less was discarded. Thus, of 52 sequences, 37 were
discarded. Those with maximum scores over 50 bits were analyzed further. The
scores and error values for the homologous sequences were recorded. The
highest score for each input sequence was noted. The probable location of the
genes of interest, their placement in the chromosome, and the specific protein
encoded by each individual gene were also taken note of. Analysis of the Preliminary Progress in Jute (Corchorus spp.) Genome Analysis 153
sequences revealed the partial DNA sequence of three hypothetical proteins,
RNA polymerase &#946 subunit-1, 18S rRNA, mitochondrial DNA directed RNA
polymerase, carboxytransferase &#946 subunit, and tRNA-Leu. As mentioned above
a known partial base sequence of putative phosphate transport ATP-binding
protein gene, partial cds was determined in C. capsularis var. CVL-1.
The early submission to GenBank (National Center for Biotechnology Information) of
the DNA base sequences of Corchorus species was of C. olitorius from Harvard
University Herbaria, MA, USA (Alverson et al. 1999). In the course of the next
four years, this group (Whitlock et al. 2003) working at University of
Massachusetts, Amherst published partial cDNA base sequences of NADH
dehydrogenase (ndhF) gene of a number of wild Corchorus species namely, C.
sidoides, C. bricchettii, C. argutus, C. siliquosus as well as fiber-yielding, species, C.
olitorius and C. capsularis. On the basis of similarity of base sequence of ndhF
gene, Whitlock et al. (2003) suggested that Oceanopapaver neocaledonicum be
transferred to Corchorus.

Another two important submissions to GenBank were by Basu et al. (2003) at
the Indian Institute of Technology, Kharagpur. Working with C. capsularis, they
determined complete cDNA sequence of caffeoyl-CoA-O-methyltransferase and
cinnamyl alcohol dehydrogenase which are two of the three genes involved in
lignin biosynthesis. Recently, Liu et al. (2005) at the Chinese Medical University,
Taiwan deposited at the GenBank, base sequence of 18S ribosomal RNA gene
of C. olitorius and C. capsularis as well as that of C. aestuans var. brevicaulis, a wild
Corchorus species from Taiwan. A phylogenetic tree was constructed comprising
several species of Corchorus and Gossypium robinsonii. Nucleotide sequences of
18s rRNA from the four plants were compared using a clustalw multiple
alignment (Multiple Sequence Alignment - CLUSTALW) by uploading a Fasta file of the nucleotide
sequences into the clustalw site. After the alignment a phylogenetic tree was
constructed using the clustalw site from the alignment score page. The NJ tree
format was used.

Fig. 2. Phylogenetic tree of selected Corchorus species and Gossypium robinsonii
based on 18S rRNA gene sequences.

The phylogenetic tree shows that Gossypium and Corchorus diverged from a
common ancestor and that all species of the Corchorus are more closely related to

Cossypium_robinsonii 154 Islam et al.
each other than to Gossypium. Furthermore, the tree shows that C. capsularis and
C. olitorius are more closely related than each of these two species to C. aestuans.
Thus the molecular data presented in this paper are in agreement with the
current taxonomic classification and the crossing relationships between different
Corchorus species as reported by the first author (Islam 1958). This phylogenetic
tree based on 18S rRNA sequences needs to be compared taking into
consideration of similar molecular characters before a consensus tree is

The joint team led by P.K. Gupta at Charan Singh University, Meerat, India
and Haseena Khan at Dhaka University, Bangladesh submitted to GenBank
DNA base sequences of SSR markers in 195 accessions of C. olitorius. These
markers were used to distinguish accessions that were cold-tolerant from
susceptible ones. The SSR sequences submitted to Genbank
(BLAST: Basic Local Alignment Search Tool) by Sharma et al (2005) were retrieved
and entered in TAIR website (TAIR - Home Page) to check for homology to
sequences in other plant species, especially the genome of Arabidopsis thaliana.
The WU-BLAST search was set to include introns and UTRs (AGI Genes
(+introns, +UTRs) (DNA)). Sequences that gave significant homology were
recorded. In some instances, a sequence showed homology to multiple
sequences in other plant taxa, in which case the sequence with the greatest score
was selected and recorded. In the case of multiple sequences having the same
score, the one with the lowest E value was selected and recorded. Any sequence
that returned matches with a maximum score of 50 bits or less was recorded as
&#8220No Hits.&#8221

Table 2. Summary of the sequence analysis of the jute SSR sequences
by Sharma et al. (2005) posted in the GenBank.

Genes Frequency Score E value
Nuclear 79 197 to 2074 0.79 to 2.7E-88
Mitochondrial 6 308 to 2310 6.9E-12 to 2.6E-122
Choloroplast 3 596 to 1550 3.1E-21 to 7.6E-65
Unidentified 107

Of the 195 sequences analyzed, 79 showed homology to nuclear DNA with
scores ranging from 197 to 2074, six showed homology to mitochondrial DNA
with scores ranging from 308 to 2310, three showed homology to chloroplast
DNA with scores ranging from 596 to 1550, and 107 were unidentified.
The DNA libraries were made using mRNA extracted from leaves of young
seedlings of the two jute species. So far the results submitted to GenBank are
partial cDNA sequences of putative phosphate transport ATP-binding protein
gene of C. capsularis var. CVL-1 (Accession No. DQ151661) and partial genomic
base sequences of 18S ribosomal RNA gene of C. olitorius var. O4 (Accession No.
DQ151662). Study of the randomly selected genomic DNA sequences of inserts Preliminary Progress in Jute (Corchorus spp.) Genome Analysis 155
indicated that they represent partial sequences of the following: RNA
polymerase &#946 subunit, chloroplast-encoded 23S ribosomal, chloroplast gene
encoding ribosomal s-12, DNA-directed RNA polymerase, mitochondrial
carboxytransferase &#946 subunit and three hypothetical proteins. These sequences
will soon be submitted to GenBank.

There should be a note of caution in deciphering genomic base sequences of
Corchorus. The record of nucleotide sequences in Corchrous in the GenBank show
that the majority of authors have not given names of the cultivar associated with
the two fiber-yielding species. It is an important omission because a large
amount of differences exist between cultivars of the two species of Corchorus in
both morphological and physiological traits that are bound to be reflected in
their DNA profiles.

The ultimate objective of the present study was to construct a complete
cDNA and genomic DNA libraries of both C. olitorius and C. capsularis so that the
genes of interest can be cloned and used to transform jute cultivars for obtaining
value-added industrial products. Since this enormous task cannot be undertaken
without adequate funding and collaboration with institutions willing to work on
a tropical crop, we have been studying the partial cDNA and genomic DNA
libraries on a limited scale. So far the genes we have isolated are either
chloroplast-, ribosomal- or mitochondrial genes or the enzymes that reside in
these structures. The main reason for obtaining genes from the above organelles
alone is that DNA has so far been isolated from young leaves that contain
numerous chloroplasts and mitochondria, and such genes are more abundant
than others. No efforts were made so far to select unique DNA fragments. Future
analysis will involve screening of the current library with probes of these
abundantly expressed genes to avoid repeated sequencing. Also, many of the
sequences are partial and full length sequences need to be isolated by cloning the
cDNA ends. Further study aims at collecting suitable material from other parts of
the plant such as young stem, bark tissue and roots to clone genes controlling
many important traits of agronomic importance including those that confer
resistance to insects, fungal and viral pathogens, and those for regulating lignin
biosynthesis. Both random sequencing and selective cloning strategies will be
employed. In addition, tissue specific promoters will be also be cloned for
potential genetic improvement. Since the entire jute genome analysis is a major
effort, an international collaboration among the scientists from India,
Bangladesh, USA and other countries is essential to help accomplish this task.
The clones and sequences should be made available for the genetic improvement
of jute to ultimately benefit the millions of jute farmers in the long run.

The authors thank Professor Haseena Khan and Samuil Haque of Dhaka
University, Bangladesh for providing jute seeds with proper phytosanitary
certificate. They thank Ray Neubauer, Thomas Garrad, Mohamad Wazni,
Lindsay Stone and Nabila Anwar who helped them with the plasmid DNA 156 Islam et al.
isolation and laboratory work. Sincere thanks to Dena Sutton and her associates
for helping with the growing of jute plants in the greenhouse of The University
of Texas at Austin. Finally they express their gratitude to the University CoOp
and the University of Texas at Austin for supporting the undergraduate research
fellowship program without which the present research could not have been


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