Information

Is a single polynucleotide necessarily and always a nucleic acid?


I am confused. I know that a DNA molecule is made of two polynucleotides.

But does each polynucleotide represent one nucleic acid? If so, a DNA molecule is made of two nucleic acids, right?

Or do the two polynucleotides (that form a molecule of DNA) make up only one nucleic acid? If so, a DNA molecule is only made of one nucleic acid, right?


There are two types of nucleic acids: 1) DNA --> which is double-stranded, so it's made of only two polynucleotides 2) RNA --> which is single-stranded so it's made of only one polynucleotide. So if we get back to my original question "Is a single polynucleotide necessarily and always a nucleic acid?" --> the answer is no, for DNA is a nucleic acid and it is made of two polynucleotides.


Your question arrangement is wrong, DNA is made up of 2 polynucleotide strand indeed. And these polynucleotides are the polymers of nucleotides, where nucleotides consist of deoxyribose sugar in case of DNA and ribose sugar in case of RNA+nitrogenous base+phosphate. Yes it is always a nucleic acid.


Strain-specified relative conformational stability of the scrapie prion protein

Studies of prion biology and diseases have elucidated several new concepts, but none was more heretical than the proposal that the biological properties that distinguish different prion strains are enciphered in the disease-causing prion protein (PrP Sc ). To explore this postulate, we examined the properties of PrP Sc from eight prion isolates that propagate in Syrian hamster (SHa). Using resistance to protease digestion as a marker for the undenatured protein, we examined the conformational stabilities of these PrP Sc molecules. All eight isolates showed sigmoidal patterns of transition from native to denatured PrP Sc as a function of increasing guanidine hydrochloride (GdnHCl) concentration. Half-maximal denaturation occurred at a mean value of 1.48 M GdnHCl for the Sc237, HY, SHa(Me7), and MT-C5 isolates, all of which have ∼75-d incubation periods a concentration of 1.08 M was found for the DY strain with a ∼170-d incubation period and ∼1.25 M for the SHa(RML) and 139H isolates with ∼180-d incubation periods. A mean value of 1.39 M GdnHCl for the Me7-H strain with a ∼320-d incubation period was found. Based on these results, the eight prion strains segregated into four distinct groups. Our results support the unorthodox proposal that distinct PrP Sc conformers encipher the biological properties of prion strains.

Many lines of evidence show that the pathogenic protein (PrP Sc ) is the sole component of the infectious prion particle and that its formation derives from the post-translational modification of the cellular isoform (PrP C Cohen and Prusiner 1998 Prusiner et al. 1998). Although the covalent structure of the two PrP isoforms appears identical (Stahl et al. 1993 ), they can be readily distinguished by their drastically different physical properties (Pan et al. 1993 ). PrP C is readily soluble in nondenaturating detergents, is rapidly digested by proteases, and is rich in α-helical structure and essentially devoid of β-sheet content. In contrast, PrP Sc is insoluble in such detergents, is resistant to proteolysis except for the N-terminal region comprising ∼67 residues, and has a high β-sheet content (Caughey et al. 1991 Gasset et al. 1993 Pan et al. 1993 Pergami et al. 1996 Safar et al. 1993 ). The protease-resistant fragment of PrP Sc has a molecular size of 27–30 kD and is designated PrP 27–30 (Prusiner et al. 1983 ). It consists of ∼142 amino acids and conveys prion infectivity. In the presence of detergent, PrP 27–30 readily polymerizes into amyloid although amyloid is neither obligatory for prion infectivity nor disease pathogenesis (Prusiner et al. 1983 , 1990 McKinley et al. 1991 ). Protein denaturants abolish prion infectivity and protease resistance while increasing solubility and immunodetection of PrP Sc (Kitamoto et al. 1987 Serban et al. 1990 Taraboulos et al. 1992 Prusiner et al. 1993 Oesch et al. 1994 Peretz et al. 1997 Safar et al. 1998 ). Thus, considerable evidence shows that prion diseases are disorders of protein conformation.

Prion strains have been shown to breed true on repeated passage in animals of the same species as by phenotypic characteristics including the clinical presentation of disease (Pattison and Millson 1961 Mastrianni et al. 1999 ), the length of the incubation period (Dickinson et al. 1968 ), the distribution of vacuolar degeneration (Fraser and Dickinson, 1968 Fraser 1979 ), and the pattern of PrP Sc deposition in the CNS (Bruce et al. 1989 Hecker et al. 1992 ). The phenomenon of prion strains has been cited frequently as evidence that an independently replicating informational molecule or genome exists within the infectious particle (Bruce and Dickinson 1987 ). To accommodate multiple strains in the absence of a nucleic acid, PrP Sc must be able to sustain separate information states within the same amino acid sequence, and PrP C must be able to faithfully acquire this information during its conversion into PrP Sc . Substantial evidence suggests that a direct interaction between PrP C and PrP Sc leads to the conversion of PrP C to PrP Sc (Prusiner et al. 1990 Horiuchi et al. 1999 ). Accordingly, different strains must maintain different templates of PrP Sc structures, and these differences at the molecular level ultimately should dictate strain properties.

Persuasive evidence that strain-specific information is enciphered in the structure of PrP Sc arose from the transmission of two different inherited human prion diseases to mice expressing a chimeric human/mouse (MHu2M) PrP transgene (Telling et al. 1996 ). In fatal familial insomnia (FFI), the protease-resistant fragment of PrP Sc after enzymatic removal of the two N-linked glycans is 19 kD, whereas that from familial Creutzfeldt-Jacob disease (CJD) and sporadic CJD is 21 kD (Monari et al. 1994 Parchi et al. 1996 ). Extracts from the brains of patients with FFI transmitted disease to Tg mice and induced formation of the 19-kD PrP Sc fragment in contrast, extracts from the brains of patients with CJD produced the 21-kD PrP Sc fragment in the same mice (Telling et al. 1996 ). These results showed that MHu2M PrP Sc can exist in two different conformations based on the sizes of the protease-resistant fragments, yet, the amino acid sequence of MHu2M PrP Sc remained invariant.

Although early comparisons of hamster prion strains did not reveal any particularly compelling biochemical differences in PrP Sc (Kascsak et al. 1986 Hecker 1992), such differences were found for two transmissible mink encephalopathy (TME) prion strains, drowsy (DY) and hyper (HY), that were transmitted to hamsters (Bessen and Marsh 1992b ). The DY strain was found to differ significantly from other known hamster prion strains in its biochemical and physical properties. Marked differences were identified by sedimentation analysis, protease sensitivity, and by the migration pattern of PrP Sc proteolytic fragments on SDS gels (Bessen and Marsh 1992b ). PrP Sc comprising the DY prions showed diminished resistance to proteinase K digestion and yielded a protease-resistant fragment of 19 kD after deglycosylation, whereas that from HY was 21 kD (Bessen and Marsh 1994 ). Notably, TME strain properties could be preserved after transmission of either the full-length or the protease-resistant fragment of PrP Sc , contending that strain characteristics are propagated by the protease-resistant core (Bessen and Marsh 1994 ).

Because most prion strains encipher PrP Sc conformers that generally yield PrP 27–30 with a polypeptide core of 21 kD after limited proteolysis, it has not been possible to use the mobility-shift assay to detect plausible differences in PrP Sc conformation in most cases (Bessen and Marsh 1994 Monari et al. 1994 Parchi et al. 1996 Telling et al. 1996 Scott et al. 1997 ). Moreover, the insolubility of PrP Sc has prevented comparative structural studies of prion strains by using high-resolution nuclear magnetic resonance and X-ray diffraction techniques. Of note, Fourier-transform infrared spectroscopy of the HY and DY strains did give different spectra (Caughey et al. 1998 ). A conformation-dependent immunoassay (CDI) was used to investigate the eight SHa prion isolates studied here by quantification of the immunoreactivity of denatured (D) and native (N) PrP Sc (Safar et al. 1998 ). The monoclonal antibody (mAb) 3F4 used in those studies recognizes a conformationally sensitive epitope (Peretz et al. 1997 ). In a plot of the D/N ratio as function of PrP Sc concentration, each isolate occupied a unique position, a result consistent with the existence of multiple discrete PrP Sc conformers that are strain-specified (Safar et al. 1998 ).

To test the hypothesis that the biological properties of prion strains are enciphered in the conformation of PrP Sc , we examined the relative conformational stability of PrP Sc derived from the SHa brains infected with eight strains. Because PrP 27–30, the protease-resistant core of PrP Sc , is infectious and can initiate the faithful propagation of strains (Prusiner et al. 1983 Bessen and Marsh 1994 ), we studied the conformational stability of this molecule. Using sensitivity to protease as a marker for the denatured state of PrP 27–30, we characterized the protein conformations of eight hamster prion isolates. We found that these eight strains could separate into four groups based on relative conformational stability and incubation period. The sigmoidal shape of the conformational transition curves shows that the unfolding of PrP Sc from a protease-resistant state to a sensitive one is a cooperative process. Our findings support the proposition that PrP Sc can adopt multiple conformations. By inference, these results lend additional support to the hypothesis that the conformation of PrP Sc enciphers the biological properties of prion strains.


2009年3月28日星期六

Why Clone Mammals?

Given that we already knew from amphibian studies in the 1960s that nuclei were pluripotent, why clone mammals? Many of the reasons are medical and commercial, and there are good reasons why these techniques were first developed by pharmaceutical companies rather than at universities. Cloning is of interest to some developmental biologists who study the relationships between the nucleus and cytoplasm during fertilization or who study aging (and the loss of totipotency that appears to accompany it), but cloned mammals are of special interest to those people concerned with protein pharmaceuticals. Protein drugs such as human insulin, protease inhibitor, and clotting factors are difficult to manufacture. Due to immunological rejection problems, the human proteins are usually much better tolerated by patients than proteins from other animals. So the problem becomes how to obtain large amounts of the human protein. One of the most efficient ways of producing these proteins is to insert the human genes encoding them into the oocyte DNA of sheep, goats, or cows. Animals containing a gene from another individual (often of a different species) a transgene are called transgenic animals. A transgenic female sheep or cow might not only contain the gene for the human protein, but might also be able to express the gene in her mammary tissue and thereby secrete the protein in her milk. Thus, shortly after the announcement of Dolly, the same laboratory announced the birth of Polly (Schnieke et al. 1997). Polly was cloned from transgenic fetal sheep fibroblasts that contained the gene for human clotting factor IX, a gene whose function is deficient in hereditary hemophilia.

Producing transgenic sheep, cows, or goats is not an efficient undertaking. Only 20% of the treated eggs survive the technique. Of these, only about 5% express the human gene. And of those transgenic animals expressing the human gene, only half are female, and only a small percentage of these actually secrete a high level of the protein into their milk. (And it often takes years for them to first produce milk). Moreover, after several years of milk production, they die, and their offspring are usually not as good at secreting the human protein as the originals. Cloning would enable pharmaceutical companies to make numerous copies of such an "elite transgenic animal," all of which should produce high yields of the human protein in their milk. The medical importance of such a technology would be great, since such proteins could become much cheaper for the patients who need them for survival. The economic incentives for cloning are therefore enormous (Meade 1997).

Cloning mammals


In 1997, Ian Wilmut announced that a sheep had been cloned from a somatic cell nucleus from an adult female sheep. This was the first time that an adult vertebrate had been successfully cloned from another adult. To do this, Wilmut and his colleagues 1997 took cells from the mammary gland of an adult (6-year-old) pregnant ewe and put them into culture.

The culture medium was formulated to keep the nuclei in these cells at the resting stage of the cell cycle (G0). They then obtained oocytes (the maturing egg cell) from a different strain of sheep and removed their nuclei. The fusion of the donor cell and the enucleated oocyte was accomplished by bringing the two cells together and sending electrical pulses through them. The electric pulses destabilized the cell membranes, allowing the cells to fuse together. Moreover, the same pulses that fused the cells activated the egg to begin development. The resulting embryos were eventually transferred into the uteri of pregnant sheep. Of the 434 sheep oocytes originally used in this experiment, only one survived: Dolly

DNA analysis confirmed that the nuclei of Dolly's cells were derived from the strain of sheep from which the donor nucleus was taken (Ashworth et al. 1998 Signer et al. 1998). Thus, it appears that the nuclei of adult somatic cells can be totipotent. No genes necessary for development have been lost or mutated in a way that would make them nonfunctional. This result has been confirmed in cows (Kato et al. 1998) and mice (Wakayama et al. 1998). In mice, somatic cell nuclei from the cumulus cells of the ovary were injected directly into enucleated oocytes. These renucleated oocytes were able to develop into mice at a frequency of 2.5% . Interestingly, nuclei from other somatic cells (such as neurons or Sertoli cells) that are similarly blocked at the G0 stage did not generate any live mice. Cumulus cell nuclei from cows have also directed the complete development of oocytes into mature cows

The split between embryology and genetics

Morgan's evidence provided a material basis for the concept of the gene. Originally, this type of genetics was seen as being part of embryology, but by the 1930s, genetics became its own discipline, developing its own vocabulary, journals, societies, favored research organisms, professorships, and rules of evidence. Hostility between embryology and genetics also emerged. Geneticists believed that the embryologists were old-fashioned and that development would be completely explained as the result of gene expression. Conversely, the embryologists regarded the geneticists as uninformed about how organisms actually developed and felt that genetics was irrelevant to embryological questions. Embryologists, such as Frank Lillie, Ross Granville Harrison (1937), Hans Spemann (1938), and Ernest E. Just (1939), claimed that there could be no genetic theory of development until at least three major challenges had been met by the geneticists:

1. Geneticists had to explain how chromosomes which were thought to be identical in every cell of the organism produce different and changing types of cell cytoplasms.

2. Geneticists had to provide evidence that genes control the early stages of embryogenesis. Almost all the genes known at the time affected the final modeling steps in development (eye color, bristle shape, wing venation in Drosophila). As Just said (quoted in Harrison 1937), embryologists were interested in how a fly forms its back, not in the number of bristles on its back.

3. Geneticists had to explain phenomena such as sex determination in certain invertebrates (and vertebrates such as reptiles), in which the environment determines sexual phenotype.

Now that the necessity of relating the data of genetics to embryology is generally recognized and the Wanderlust of geneticists is beginning to urge them in our direction, it may not be inappropriate to point out a danger of this threatened invasion. The prestige of success enjoyed by the gene theory might easily become a hindrance to the understanding of development by directing our attention solely to the genom, whereas cell movements, differentiation, and in fact all of developmental processes are actually effected by cytoplasm. Already we have theories that refer the processes of development to gene action and regard the whole performance as no more than the realization of the potencies of genes. Such theories are altogether too one-sided.

Until geneticists could demonstrate the existence of inherited variants during early development, and until geneticists had a well-documented theory for how the same chromosomes could produce different cell types, embryologists generally felt no need to ground their science in gene action.

Nucleus or cytoplasm: Which controls heredity?

It is in Mendel's term, however, that we see how closely intertwined were the concepts of inheritance and development in the nineteenth century. Mendel's observations, however, did not indicate where these hereditary elements existed in the cell, or how they came to be expressed. The gene theory that was to become the cornerstone of modern genetics originated from a controversy within the field of physiological embryology. In the late 1800s, a group of scientists began to study the mechanisms by which fertilized eggs give rise to adult organisms. Two young American embryologists, Edmund Beecher Wilson and Thomas Hunt Morgan, became part of this group of "physiological embryologists," and each became a partisan in the controversy over which of the two compartments of the fertilized egg the nucleus or the cytoplasm controls inheritance. Morgan allied himself with those embryologists who thought the control of development lay within the cytoplasm, while Wilson allied himself with Theodor Boveri, one of the biologists who felt that the nucleus contained the instructions for development. In fact, Wilson 1896 declared that the processes of meiosis, mitosis, fertilization, and unicellular regeneration (only from the fragment containing the nucleus) "converge to the conclusion that the chromatin is the most essential element in development."* He did not shrink from the consequences of this belief. Years before the rediscovery of Mendel or the gene theory, Wilson 1895 noted, "Now, chromatin is known to be closely similar, if not identical with, a substance known as nuclein . which analysis shows to be a tolerably definite chemical composed of a nucleic acid (a complex organic acid rich in phosphorus) and albumin. And thus we reach the remarkable conclusion that inheritance may, perhaps, be effected by the physical transmission of a particular chemical compound from parent to offspring."

Some of the major support for the chromosomal hypothesis of inheritance was coming from the embryological studies of Theodor Boveri, a researcher at the Naples Zoological Station. Boveri fertilized sea urchin eggs with large concentrations of their sperm and obtained eggs that had been fertilized by two sperm. At first cleavage, these eggs formed four mitotic poles and divided into four cells instead of two. Boveri then separated the blastomeres and demonstrated that each cell developed abnormally, and in a different way, as a result of each of the cells having different types of chromosomes. Thus, Boveri claimed that each chromosome had an individual nature and controlled different vital processes.

Adding to Boveri's evidence, E. B. Wilson and Nettie Stevens demonstrated a critical correlation between nuclear chromosomes and organismal development: XO or XY embryos became male XX embryos became female. Here was a nuclear property that correlated with development. Eventually, Morgan began to obtain mutations that correlated with sex and with the X chromosome, and he began to view the genes as being physically linked to one another on the chromosomes. The embryologist Morgan had shown that nuclear chromosomes are responsible for the development of inherited characters.


Watch the video: Finding alien life: Christoph Adami at TEDxUIUC (January 2022).