Is the CMV promoter active in yeast?

I wish to transfect yeast and want to control my efficiency. So I thought to use pEGFP-N1 as control. The EGFP is driven by a CMV promoter. Can yeast read CMV promoters. Thanks Hermann

Yes (reference)

While I found your question interesting this was pretty much the first hit when I typed "CMV yeast" in google scholar. You should really look there first before you ask!

The CMV early enhancer/chicken β actin (CAG) promoter can be used to drive transgene expression during the differentiation of murine embryonic stem cells into vascular progenitors

Mouse embryonic stem cells cultured in vitro have the ability to differentiate into cells of the three germ layers as well as germ cells. The differentiation mimics early developmental events, including vasculogenesis and early angiogenesis and several differentiation systems are being used to identify factors that are important during the formation of the vascular system. Embryonic stem cells are difficult to transfect, while downregulation of promoter activity upon selection of stable transfectants has been reported, rendering the study of proteins by overexpression difficult.


CCE mouse embryonic stem cells were differentiated on collagen type IV for 4–5 days, Flk1 + mesodermal cells were sorted and replated either on collagen type IV in the presence of VEGFA to give rise to endothelial cells and smooth muscle cells or in collagen type I gels for the formation of vascular tubes. The activity of the CMV and β-actin promoters was downregulated during selection of stable transfectants and during differentiation to the Flk1 stage, while the CMV immediate enhancer/β-actin promoter in the pCAGIPuro-GFP vector led to 100% of stably transfected undifferentiated and differentiated cells expressing GFP. To further test this system we expressed syndecan-2 and -4 in these cells and demonstrated high levels of transgene expression in both undifferentiated cells and cells differentiated to the Flk1 stage.


Vectors containing the CAG promoter offer a valuable tool for the long term expression of transgenes during stem cell differentiation towards mesoderm, while the CMV and β-actin promoters lead to very poor transgene expression during this process.


One of the characteristics of synthetic biology is de novo design and synthesis of biological functional parts and devices. Promoters are especially important for controlling the regulation-modes and strengths of gene expression, generating proper enzyme amounts to optimize cellular metabolism. Yeast is one of the most frequently used chassis in the research of synthetic biology, presenting excellent performance as cell factories to produce varied valuable biochemicals [1,2,3]. Appropriate promoter strengths drastically affected efficiency of heterologous synthetic pathways in yeast and consequent product compositions [4,5,6,7]. Recent works have established the modular architecture of yeast promoters, such as in baker’s yeast Saccharomyces cerevisiae, methylotrophic Pichia pastoris and oleaginous Yarrowia lipolytica [8,9,10,11,12,13]. Redden and Alper’s work is especially outstanding as they defined and proved a design mode of minimal yeast promoter that conserved high levels of expression with almost 80% reduction in size [8]. The minimal modular yeast promoter consists of several basic parts in order, namely, hybrid upstream activating sequence (UAS), neutral AT-rich spacer, TATA-box, N30 core promoter, and transcriptional starting site (TSS). This work offers a great chance for designing artificial promoters with more generalized sequences.

Among these modular parts, UAS, AT-rich spacer, TATA-box and TSS have distinctive conservative features as only limited number of natural or artificial sequences would preserve or enhance promoter’s strength but most engineered sequences drastically decrease promoter’s strength [6, 8,9,10,11,12,13,14,15,16]. By contrast, the core promoter sequence downstream of the TATA box influenced the gliding speed of RNA polymerase II before the mRNA was generated, also determining maximal promoter activity. This region allows more space of artificial design but also has its own limitedness, as the thoroughly randomized sequences will overwhelm effective sequence that enhance promoter’s strength. Portela and colleagues’ recent work made a remarkable chance to design universal mode of core promoter that could be used in different yeast species [17]. There still leaves a question whether this region can be replaced by a de novo designed artificial sequence.

From another angle, we need this kind of de novo designed artificial promoters as barcodes to mark gene expression at genome level to research the genome rearrangement [18, 19]. The artificial barcodes of promoters will further assist the analysis of synthetic genomic evolution. Considering this point, we plan to construct a series of artificial core promoter sequences and characterize their performances to get some kind of rule. In the present study, we constructed artificial core promoters in Y. lipolytica, which was engineered for producing valuable chemicals as fatty acid derivatives, organic acids, terpenoids and sterols [20,21,22]. We chose an existing object that the artificial promoters might enhance the expression levels of CrtY enzyme to get higher beta-carotene production form the substrate lycopene (Fig. 1). We could rapidly screen desirable promoters contained in the yeast colonies with different colors of red–orange–yellow that presented different lycopene–carotene compositions (Fig. 1). The colonies with lycopene production of 1.2 mg/g DCW and much higher 25 mg/g would present highly differentiate color-spectrum. We designed two series of N30 core promoters. The first series were located downstream of a natural promoter PEXP1 and replaced the original 55 bases between TATA-box and TSS in PEXP1. The second series replaced the original region of 61 bases between TATA-box and the TSS in another natural promoter PGPD. Both promoters were commonly used and owned obviously stronger strengths than other promoters. Since N30 was large library, we here focused to test the influences of T-rich modules and G/C-rich modules on core promoter’s strength.

Concept of screening artificial core promoters assisted by yeast colony-color spectrum. The engineered artificial promoters changed the expression level of CrtY enzyme and tuned the lycopene–carotene composition, leading to variable yeast colony color distribution. In the strain with low lycopene production, we observed two colors as the original light red and new light yellow. In the higher-lycopene-production strain, we observed three colors as the original red and new orange and yellow


A modified retroviral system capable of gene knockdown and expression

Mouse primary macrophages are hard to be transfected or nucleofected. However, they can be infected with retroviruses. We compared several commercialized retroviral vectors and found that pSuper-Retro-puro from Oligoengine worked best for virus production. It has at least two confirmed features, one is the shRNA expression driven by H1 promoter, the other is the expression of puromycin drug resistance marker driven by PKG promoter. However, H1 promoter may not function with the same strength as a U6 promoter for shRNA expression (16) , and this vector lacks the cloning sites for exogenous gene expression. We therefore modified this vector into a new retroviral vector, capable of gene knockdown and expression while reserving the puromycin resistance expression function. As illustrated in Figure 1A , we removed the H1 promoter in pSuperRetro-puro and reserved the multiple cloning sites (MCS1) for cloning of U6 promoter driving shRNA expression. Human UbiC promoter has been shown to be constitutively active in a variety of cells and tissues (27) , and was selected to drive the expression of an exogenous gene and puromycin resistance gene separated by SV40 promoter. MCS2 between UbiC and SV40 allows the cloning of exogenous gene. We derived vectors with C-terminal Flag-His6 (FH) to allow for immunoblot or immunoprecipitation of the expressed protein, and named this new vector as pFRRu. The other vector pFRRg has the same design strategy, but UbiC promoter was replaced with PKG promoter ( Figure 1B ). With these new retroviral vectors we have transduced a variety of mouse cell types (e.g., epithelial cells, keratinocytes, fibroblasts, and hematopoietic cells) as well as primary cells [e.g., mouse embryonic fibroblasts and bone marrow-derived macrophages (BMDMs)]. The efficiency of transduction varies with cell type and the virus titer applied. However, following puromycin selection, all the residual cells were virally transduced. In this study, the viral titer we applied led to 30%-60% transduction efficiency for macrophages, avoiding multiple entries of viral transcripts into one cell.

A modified retroviral system with gene knockdown and exogenous gene expression functions. A. Map of the modified retroviral vector with UbiC promoter. pFRRu was generated as described in Materials and Methods. There are two multiple cloning sites: 5′-end of UbiC has the multiple cloning sites for shRNA expression cassette (MCS1), and 3′-end of UbiC has the multiple cloning sites for exogenous gene expression (MCS2) containing an in-frame Flag-His6 (FH) tag. Puromycin (puro) resistance expression was reserved with SV40 promoter. B. The modified retroviral vector with PKG promoter. UbiC promoter as described above was replaced with PKG promoter.

Inhibition of U6 promoter activity by divergent promoter arrangement of U6 and UbiC

Usually three promoter arrangements may lead to TI, namely convergent promoters, tandem promoters and overlapping (divergent) promoters (26) . Since convergent promoter arrangement for U6 and other promoters is rare in viral vectors, we focused on tandem and divergent promoter arrangements to explore which arrangements led to significant impairment to U6 promoter activity. We constructed U6 promoter driving mCIN85 shRNA expression cassette and inserted this expression cassette in MCS1 of pFRRu in different orientations. One was divergent promoter arrangement in which U6 and UbiC were in opposite direction (pU6sh-pUbiC divergent), and the other was tandem in the same direction as UbiC (pU6sh-pUbiC-tandem). We also generated a luciferase shRNA expression vector as a non-specific shRNA control and a pFRRrfp-U6-mCIN85 shRNA as a control to minimize promoter interaction by replacing UbiC promoter with a non-promoter DNA fragment ( Figure 2A ). These vectors were introduced into Plat E cells to produce viruses. BMDMs were transduced with respective virues and subsequently selected with puromycin to remove the non-transduced cells. After four days of selection the remaining cells were lysed for protein extraction. Western blots were carried out using rabbit anti-CIN85 antibody ( Figure 2B ). Relative U6 promoter activity was determined using endogenous mCIN85 protein level normalized against the α-tubulin level as the loading control. As shown in Figure 2C , the arrangement of U6 promoter relative to UbiC promoter in the vector had a significant impact on U6 promoter activity. Tandem arrangement maintained much higher U6 promoter activity than divergent arrangement. This suggested that U6 promoter activity can be regulated by TI in a promoter arrangement-dependent manner.

Divergent promoter arrangement of U6 and UbiC inhibits U6 promoter activity. A. Diagram of U6 and UbiC promoter arrangements. mCIN85 shRNA expression cassette was constructed as described in Materials and Methods, and was inserted into pFRRu to form divergent and tandem promoter arrangements. Minimal interfered U6 promoter activity control was set by replacing the UbiC promoter with a non-promoter cDNA fragment of RFP. B. Residual endogenous mCIN85 protein level to reflect U6 promoter activity. Mouse BMDMs were transduced with retroviruses produced with different vectors as indicated. Four days after viral transduction and drug selection, residual cells were collected and lysed. Western blots were performed using purified rabbit anti-CIN85 as primary antibody. C. Normalized mCIN85 knocking down efficiency reflecting relative U6 promoter activity. Sample sequences are as indicted in Panel B. Values are statistics from three independent experiments.

CMV enhancer negatively impacts U6 promoter activity in the presence of UbiC promoter

Previous studies have shown that the CMV enhancer has a positive effect on U6 promoter activity (23) . However, the effect of the CMV enhancer on U6 promoter activity in the presence of TI is unknown. To answer this question, we placed a CMV enhancer between U6 and UbiC promoters in both promoter arrangements by fusing the enhancer to the upstream of UbiC or U6 promoter ( Figure 3A ). We then tested the U6 promoter activity. To our surprise, instead of enhancing U6 promoter activity, CMV enhancer in all four configurations significantly strengthened the UbiC inhibitory effect on U6 promoter activity in both promoter arrangements ( Figure 3B and C ). However, the level of inhibition varied with the promoter arrangements. Fusing the CMV enhancer upstream of U6 and keeping tandem arrangement of the promoters gave relatively less inhibition, while the divergent configuration gave the highest inhibition. This result indicates that the CMV enhancer can boost TI and significantly inhibit U6 promoter activity in the presence of UbiC promoter in either promoter arrangements.

Negative impact of UbiC on U6 promoter activity enhanced in the presence of CMV enhancer. Legends in Figure 2 was followed except that CMV enhancer was placed between U6 and UbiC promoter or fused to the upstream of U6 as plotted. A. Diagram of U6 promoter, CMV enhancer, and UbiC promoter arrangements. CMV enhancer was fused to the upstream of either U6 promoter or UbiC promoter forming divergent or tandem arrangements as indicated. B. Western blot of mCIN85 to reflect the residual mCIN85 left in the cells. Samples were generated using different virus transduction as indicated. C. Relative U6 promoter activity after normalization against α-tubulin. Values are statistics from three independent experiments.

Regulation of U6 promoter activity by TI is promoter-specific

TI is often originated from asymmetric strength of two closely arranged promoters, the stronger promoter reduces the activity of the weaker one (26) . UbiC promoter is ubiquitously active in a variety of cells and is a relatively strong promoter (27) . We next asked whether U6 promoter activity can be maintained if we replace the UbiC promoter with a weaker PKG promoter to balance the previously asymmetric strength. We constructed similar viral vectors in both U6 promoter arrangements with PKG promoter as shown in Figure 4A and tested U6 promoter activity in transduced BMDMs. As expected, no significant inhibition of U6 promoter activity was observed in either arrangement ( Figure 4B and C ). This result suggests that regulation of U6 promoter activity by TI is promoter-specific. Balancing the strength of the two adjacent promoters is the key. PKG, a weaker promoter, has minimum TI effect on U6 promoter activity.

U6 promoter activity response to TI is promoter-specific. Legends in Figure 2 were followed except that UbiC promoter was replaced with PKG promoter. A. Diagram of U6 and PKG promoter arrangements. B. Residual endogenous mCIN85 protein level to reflect U6 promoter activity. C. Relative U6 promoter activity after normalization against α-tubulin. Values are statistics from three independent experiments.

Visualization of gene activity in living cells

Chromatin structure is thought to play a critical role in gene expression. Using the lac operator/repressor system and two colour variants of green fluorescent protein (GFP), we developed a system to visualize a gene and its protein product directly in living cells, allowing us to examine the spatial organization and timing of gene expression in vivo. Dynamic morphological changes in chromatin structure, from a condensed to an open structure, were observed upon gene activation, and targeting of the gene product, cyan fluorescent protein (CFP) reporter to peroxisomes was visualized directly in living cells. We found that the integrated gene locus was surrounded by a promyelocytic leukaemia (PML) nuclear body. The association was transcription independent but was dependent upon the direct in vivo binding of specific proteins (EYFP/lac repressor, tetracycline receptor/VP16 transactivator) to the locus. The ability to visualize gene expression directly in living cells provides a powerful system with which to study the dynamics of nuclear events such as transcription, RNA processing and DNA repair.


Mitochondrial targeting of C-anchored outer membrane proteins has not yet been thoroughly investigated. As the first step toward clarifying their targeting mechanisms, we characterized the mitochondrial targeting signal by using the GFP-Tom5 fusions as a model. Because fluorescent GFP fusions expressed in vivo possess a “tight fold” and the Tom5 peptide attached to the C-terminal of GFP can be as short as 50 residues, the C-terminal of Tom5 should always be exposed to the surface of the fluorescent active molecule. Thus, the fluorescence represents correctly folded proteins and, therefore, the present assay discounts nonspecific association of the unfolded proteins to various organelles. Our results demonstrated that the TMS with 18–20 hydrophobic residues and positive charges in the following C-segment are both important determinants for mitochondrial targeting. The importance of the C-segment as the mitochondrial targeting signal was most clearly shown by the experiment in Figure 2 the C-segment of Tom5 transplanted to the C-terminal of cytochromeb5, directed otherwise ER-targeted protein to the mitochondria, or vice versa. On reduction of the number of positive charges in the C-segment, the mutants gradually lost membrane specificity and were distributed not only to mitochondria but to the ER, indicating that at least three basic amino acids at the C-segment are required for the specific targeting of Tom5 to the mitochondria.

The membrane specificity was also lost when the TMS was elongated, even although the C-segment remained intact (Figure 4). In view of the report that hydrophobic forces drive spontaneous membrane insertion (Enoch et al., 1979 Rachubinski et al., 1980Anderson et al., 1983), we expect that the elongated TMS functions as a dominant, nonspecific membrane insertion signal (Blobel, 1980) by its enhanced affinity with the lipid bilayer, and mutated proteins were distributed throughout the membranes, including mitochondria and ER, although whether these constructs also localized to the other membrane systems remains to be determined.

Taken together, three basic amino acid residues positioned at the C terminus of the TMS with an appropriate length functioned as the mitochondrial targeting signal. This structural feature is also conserved in mitochondrial VAMP-1B (Figure 1). When both arginine residues or all three residues were changed to threonine, the mutants were transported to the membranes of the secretory organelles via the ER (Isenmann and Wattenberg, 1998). The present study demonstrated that length, rather than hydrophobicity, is the major determinant for TMS function [Figure 4, TM(H)]. In support of this, VAMP-1B has a TMS of 17 residues with a mean hydrophobicity of 3.20 nevertheless, it localized in the mitochondria. Therefore, it seems to be the length, rather than the hydrophobicity, that determines targeting to the mitochondrial outer membrane the length might be required to adapt to the thickness of the lipid bilayer of the mitochondrial outer membrane.

Insertion of five serine residues between the TMS and C-segment severely interfered with mitochondrial targeting of Tom5, whereas their addition to the C-terminal end was ineffective. Thus, the distance between the hydrophobic TMS and the basic C-segment is a critical factor for mitochondrial targeting. This observation is consistent with the previous observation that the arginine locating just after the TMS in OMb is more critical for the mitochondrial targeting than another arginine located in the distal C-terminal side (Kuroda and Ito, 1998 Figure 1).

There was a difference between the former and the latter half of the TMS in the sensitivity to the introduced mutation. A single amino acid deletion within the latter half of the TMS (40 M-45V) interfered with the mitochondrial targeting function more strongly than did a single amino acid deletion within the former half of the TMS (Figure 6). These results again indicated that the TMS and the basic C-segment should be within a suitable distance or context. Taken together, some factors in the cytosol might recognize these structural features and direct them to the mitochondrial outer membrane.

The C-terminal domain of Tom5, consisting of the TMS and C-segment, when transplanted to the N-terminal of GFP, functioned as an ER-targeting signal, probably as the signal anchor (Kida et al., 2000). Therefore, these segments must be located at the C terminus to be recognized correctly as the mitochondrial targeting signal the structural requirements of the mitochondrial targeting signals for N-anchored and C-anchored proteins are clearly distinct. Considering that the large ribosomal subunit houses the extended peptide of 39 residues (Blobel and Sabatini, 1970), the mitochondrial targeting signal of Tom5 thus characterized is almost completely protected within the large ribosomal subunit. Thus, the targeting reaction should proceed during posttranslational processing, which probably evades recognition by SRP, because recognition by SRP of the signal peptide occurs on the ribosome-nascent chain complex cotranslationally (Walter and Johnson, 1994).

The structural characteristics of the signal thus defined using yeast Tom5 were well conserved in mammalian C-tail anchor proteins Vamp1B (Isenmann and Wattenberg, 1998 discussed above) and OMP25. The membrane anchored GFP-Tom5 constructs and GFP-OMP25 were present in the dispersed state in the outer membranes and not integrated into the TOM complex. Therefore, GFP-Tom5 can be regarded as the model representing general C-tail anchor proteins that are not restricted to the TOM import machinery, but dispersed eventually into the lipid bilayers. These proteins seemed to be targeted through an identical pathway because they were imported into mitochondria that had been treated with trypsin to remove the outer membrane import receptors rTOM70, rTOM20, rTOM22, and OM37 (our unpublished data), although the involvement of the channel component rTOM40 remains to be analyzed.

The heterologous assay system with yeast Tom5 enabled us to distinguish between targeting and membrane integration steps in mammalian mitochondria. Wild-type GFP-Tom5 expressed in COS-7 cells was correctly targeted to mitochondria as observed under confocal microscopy, but was inefficiently integrated into the mitochondrial membrane, whereas the same construct expressed in yeast cells was efficiently integrated into the mitochondrial membrane. On increase of the hydrophobicity of the TMS, the fusion construct TM(H) was now firmly anchored to the mitochondrial membrane. These results suggest that the characteristics of the targeting signal of the C-tail anchor proteins are distinct between yeast and mammals. In fact, basic amino acid residues in the C-segment of GFP-Tom5 were not required for correct mitochondrial targeting and insertion of GFP-Tom5 in yeast (Horie, Sakaguchi, and Mihara, unpublished data). Characterization of the mitochondrial targeting signal of the C-tail anchor proteins in yeast is in progress.

How are these features of the signal recognized in the cytoplasm during posttranslational targeting? The nascent polypeptide associated complex (NAC), which has been characterized as the heterodimeric, ribosome-associated chaperone that prevents promiscuous interaction between SRP and the nascent polypeptides destined for cellular compartments other than the secretory pathway (Wiedmann et al., 1994), is involved in targeting of preproteins to the mitochondria in yeast (George et al., 1998Fünfschilling and Rospert, 1999). Yeast Δegd2mutants, lacking the NAC function, accumulate GFP-Tom22 and GFP-Bcl2 in the cytosol (Egan et al., 1999). The NAC seems to function as a general chaperone to maintain the organelle-targeting competence of the precursor in vivo. The present findings that the TMS and the basic C-segment must be within an appropriate context or distance for mitochondrial targeting function suggest that some factors in addition to the NAC that specifically recognize these features and stabilize the hydrophobic nascent protein in the cytosol, participate in the targeting.


Within the Herpesviridae, CMV belongs to the Betaherpesvirinae subfamily, which also includes the genera Muromegalovirus and Roseolovirus (human herpesvirus 6 and human betaherpesvirus 7). [7] It is also related to other herpesviruses within the Alphaherpesvirinae subfamily, which includes herpes simplex viruses 1 and 2 and varicella-zoster virus, and the Gammaherpesvirinae subfamily, which includes Epstein–Barr virus and Kaposi's sarcoma-associated herpesvirus. [6]

Several species of Cytomegalovirus have been identified and classified for different mammals. [7] The most studied is human cytomegalovirus (HCMV), which is also known as human betaherpesvirus 5 (HHV-5). Other primate CMV species include chimpanzee cytomegalovirus (CCMV) that infects chimpanzees and orangutans, and simian cytomegalovirus (SCCMV) and Rhesus cytomegalovirus (RhCMV) that infect macaques CCMV is known as both panine beta herpesvirus 2 (PaHV-2) and pongine betaherpesvirus 4 (PoHV-4). [8] SCCMV is called cercopithecine betaherpesvirus 5 (CeHV-5) [9] and RhCMV, Cercopithecine betaherpesvirus 8 (CeHV-8). [10] A further two viruses found in the night monkey are tentatively placed in the genus Cytomegalovirus, and are called herpesvirus aotus 1 and herpesvirus aotus 3. Rodents also have viruses previously called cytomegaloviruses that are now reclassified under the genus Muromegalovirus this genus contains mouse cytomegalovirus (MCMV) is also known as murid betaherpesvirus 1 (MuHV-1) and the closely related Murid betaherpesvirus 2 (MuHV-2) that is found in rats. [11]

Species Edit

The genus consists of these 11 species: [5]

  • Aotine betaherpesvirus 1
  • Cebine betaherpesvirus 1
  • Cercopithecine betaherpesvirus 5
  • Human betaherpesvirus 5
  • Macacine betaherpesvirus 3
  • Macacine betaherpesvirus 8
  • Mandrilline betaherpesvirus 1
  • Panine betaherpesvirus 2
  • Papiine betaherpesvirus 3
  • Papiine betaherpesvirus 4
  • Saimiriine betaherpesvirus 4

Viruses in Cytomegalovirus are enveloped, with icosahedral, spherical to pleomorphic, and round geometries, and T=16 symmetry. The diameter is around 150–200 nm. Genomes are linear and nonsegmented, around 200 kb in length. [4]

Genus Structure Symmetry Capsid Genomic arrangement Genomic segmentation
Cytomegalovirus Spherical pleomorphic T=16 Enveloped Linear Monopartite

Herpesviruses have some of the largest genomes among human viruses, often encoding hundreds of proteins. For instance, the double‑stranded DNA (dsDNA) genome of wild-type HCMV strains has a size of around 235 kb and encodes at least 208 proteins. It is thus longer than all other human herpesviruses and one of the longest genomes of all human viruses in general. It has the characteristic herpesvirus class E genome architecture, consisting of two unique regions (unique long UL and unique short US), both flanked by a pair of inverted repeats (terminal/internal repeat long TRL/IRL and internal/terminal repeat short IRS/TRS). Both sets of repeats share a region of a few hundred bps, the so-called “a sequence” the other regions of the repeats are sometimes referred to as “b sequence” and “c sequence”. [12]

Viral replication is nuclear and lysogenic. Entry into the host cell is achieved by attachment of the viral glycoproteins to host receptors, which mediates endocytosis. Replication follows the dsDNA bidirectional replication model. DNA templated transcription, with some alternative splicing mechanism is the method of transcription. Translation takes place by leaky scanning. The virus exits the host cell by nuclear egress, and budding. Humans and monkeys serve as the natural hosts. Transmission routes are dependent on coming into contact with bodily fluids (such as saliva, urine, and genital secretions) from an infected individual. [4] [13]

Genus Host details Tissue tropism Entry details Release details Replication site Assembly site Transmission
Cytomegalovirus humans monkeys Epithelial mucosa Glycoproteins Budding Nucleus Nucleus Urine saliva

All herpesviruses share a characteristic ability to remain latent within the body over long periods. Although they may be found throughout the body, CMV infections are frequently associated with the salivary glands in humans and other mammals. [7]

The CMV promoter is commonly included in vectors used in genetic engineering work conducted in mammalian cells, as it is a strong promoter and drives constitutive expression of genes under its control. [14]

Cytomegalovirus was first observed by German pathologist Hugo Ribbert in 1881 when he noticed enlarged cells with enlarged nuclei present in the cells of an infant. [15] Years later, between 1956 and 1957, Thomas Huckle Weller together with Smith and Rowe independently isolated the virus, known thereafter as “cytomegalovirus”. [16] In 1990, the first draft of human cytomegalovirus genome was published, [17] the biggest contiguous genome sequenced at that time. [18]


We have investigated the transcriptional activity of human cytomegalovirus, herpes thymidine kinase, human chorionic gonadotropin α, somatostatin, immunoglobulin κ chain, α crystallin, albumin and interferon-β promoters in the fission yeast Schizosaccharomyces pombe. Among these, the human cytomegalovirus, human chorionic gonadotropin α, and somatostatin promoters were found to be very active, approximately 11-, 9-, and 0.9-fold as active as the SV40 early promoter, respectively. The remainder of the promoters studied were weak, having only 10–20% of the SV40 promoter activity. Primer extension analysis showed that the strong promoters initiated transcription in S. pombe at the same sites as in mammalian cells, indicating the high similarity between both transcriptional systems.

Spermatid-specific promoter of the SP-10 gene functions as an insulator in somatic cells

Spermatid differentiation markers such as the acrosomal protein SP-10 display remarkable testis- and germ cell-restricted gene expression. However, little is known about the mechanisms that prevent their expression in somatic tissues. We have previously noted that the -408/+28 or the -266/+28 promoter of SP-10 directed strictly spermatid-specific transcription in transgenic mice, Biol. Reprod. 61, 1256-1266). Lack of ectopic expression in these mouse lines implied that the SP-10 promoter might have protected the transgene from the influence of neighboring enhancers. The present study tested this directly by performing enhancer-blocking assays. In transiently transfected COS cells, the -408/-92 SP-10 promoter, but not stuffer DNA, blocked the transcriptional activity of a heterologous enhancer (CMV) in a position- and orientation-dependent manner. In transgenic mice, despite integration adjacent to the pan-active CMV enhancer, the -408/+28 promoter maintained spermatid-specificity and no ectopic expression of the transgene resulted. Enhancer blocking is a characteristic feature of insulators. Our results show that the SP-10 proximal promoter, which activates transcription in spermatids, functions as an insulator in somatic cells. Insulator activity mapped to the -186/-135 region and mutation of two ACACAC motifs compromised the insulator function. In conclusion, the evolutionarily conserved SP-10 insulator is novel and is the first one shown to regulate transcription of a germ cell differentiation marker.

The Molecular Basis of Endocrinology


RNA polymerase II binds downstream of the TATA box and initiates transcription of the RNA copy of one strand of the gene. Transcription continues some way downstream of the end of the gene and the transcript is processed while being exported from the nucleus. The 5′–end is modified (capped), introns are removed (splicing) and the 3′–end is trimmed and tailed with 5- to 25-adenosine residues (polyadenylation).

RNA processing

The splicing process involves a complex series of reactions catalysed by a set of small nuclear ribonuclear protein particles (SNRPS pronounced snurps). These recognize sequences at the ends of introns enabling the precise removal of the intron sequence with reconnection of the ends of the two exons. The 5′–sequence is: Exon NNNNN^ guaagunnnnn Intron whereas the 3′–sequence is Intron nnnnnnannnnn(c/u)ncaĝNNNNN Exon. The excised intron forms a lariat in the process ( Figure 2.2) .

Figure 2.2 . Transcription and RNA processing: the primary transcript starts close to the site of RNA polymerase binding and extends beyond the polyadenylation signal. The transcript is cleaved and polyadenylated close to the signal AAUAAA the 5′-end is modified by addition of a deoxyguanosine residue ‘capping’. 5′ untranslated 3′ untranslated

Mutations of the sequences shown are a common cause of genetic disease leading to abnormal processed RNA transcripts which cannot be translated and which are often unstable leading to low levels of RNA.

The splicing reaction may proceed differently in different tissues leading to different mature RNAs and hence different protein products being produced from the same gene in a process known as alternative splicing, the best known example in endocrinology being the calcitonin/CGRP gene in which calcitonin lies on exon 4 and CGRP (calcitonin gene-related peptide) lies on exon 5. In C cells exon 4 is included in the mRNA and its poly(A) addition site is used thus removing CGRP from the mature mRNA, whereas in the nervous system exon 4 is spliced out leaving exon 5 in the mature mRNA which allows translation of CGRP ( Figure 2.3 ).

Figure 2.3 . Calcitonin gene alternate splicing: the calcitonin gene contains six exons exon 4 codes for calcitonin and contains a polyadenylation signal exon 5 codes for CGRP (calcitonin gene-related peptide) and the next polyadenylation site is in exon 6. Splicing in C cells produces an mRNA containing the first four exons and excludes exons 5 and 6, whereas in nerves exon 4 is excluded by a different tissue–specific splicing pattern, giving an mRNA containing exons 1,2,3,5 (CGRP) and 6. The different mRNAs are translated giving calcitonin or CGRP in association with different flanking peptides

Redox Cell Biology and Genetics Part B

Klaus Felix , . Siegfried Janz , in Methods in Enzymology , 2002

Shuttle Vector and Principle of Assay

In the plasmid-based shuttle vector pUR288, lacZ functions as both target and reporter gene of mutagenesis. The 5346-bp vector contains in addition to lacZ-coding sequences the 35-bp binding site for the LacI repressor, lacO, the binding site for CRP (cAMP receptor protein, which facilitates transcription of lacZ by stimulating the formation of an active promoter complex), an origin of replication (ori), and an ampicillin resistance gene ( Fig. 4A ). The principle of the pUR288 mutagenesis assay is illustrated in Fig. 4B . Briefly, shuttle vectors are excised from the transgenic concatemer by restriction with HindIII. Linearized plasmids are then separated from bulk genomic DNA with the help of magnetic beads that are coated with a LacI fusion protein that can bind to the lacO sequence of the plasmid. 4 Elution from the beads is achieved by adding isopropyl-β-D-thiogalactopyranoside (IPTG), an inactivator of LacI that induces an affinity decreasing conformational change in the protein for lacO. Plasmids are circularized at the cohesive HindIII sites by ligation with T4 ligase and then electroporated into E. coli that is (1) deficient in β-Gal (lacZ − ), (2) galactose intolerant due to the absence of galactose epimerase (galE − ), and (3) restriction negative to prevent the degradation of incoming methylated plasmid DNA. The galE − mutation is key 12 because it allows for the positive selection of lacZ − mutants in the presence of the lactose analog P-Gal (phenyl-β-D galactoside), a substrate for β-Gal. LacZ − mutants are unable to cleave P-Gal in contrast, wild-type LacZ + cells are able to cleave it, and thereby release galactose. Galactose is converted to UDP-galactoside, which cannot be further metabolized on the galE − background instead, it is accumulated intra-cellularly to toxic and eventually lytic concentrations. Thus, whereas LacZ + cells are prevented from growth on P-Gal-supplemented agar plates, LacZ − cells are not. To determine the rescue efficiency of plasmids from genomic DNA, a small aliquot (usually 2 ΜL) of a 2-ml suspension of pUR288-transfected E. coli is plated on a titer plate that has been supplemented with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal). X-Gal is cleaved by β-Gal, which produces a blue halo around growing LacZ + colonies. Note that LacZ − mutants will be missed on the dilution plate (no blue halo), but this is negligible because the ratio between LacZ + to LacZ − colonies is on the order of 10 4 :1. Note further that the cleavage of X-Gal releases galactose, which is converted to the same toxic UDP-galactoside derived from P-Gal however, the amounts of galactose liberated from X-Gal are small and therefore compatible with the growth of LacZ + colonies [the molar concentration of X-Gal (183 μM 75 μg/ml) is 64 times lower than that of P-Gal (11.7 mM, 3 mg/ml)]. The remaining part of the suspension (1998 μl) is plated on a single P-Gal plate to select for mutants. Mutants grow as small, red, formazan-stained colonies in the presence of a tetrazolium salt that should be added for improved visibility of the sometimes tiny colonies. The mutant frequency is calculated as the ratio of mutants to nonmutants that is, the number of colonies on the P-Gal selection plate to the number of colonies (×1000) on the X-Gal titer plate. To characterize the mutational spectrum, mutant colonies can be picked and used directly as templates in long-range PCR amplifications of the entire lacZ gene. The PCR fragments are useful for restriction analysis and DNA sequencing. Plasmid DNA minipreparations are equally useful if it is decided not to use PCR for template preparation. Excellent reviews of the pUR288 assay including detailed protocols and sections on troubleshooting are available. 13–15 More specialized articles on the detection of so-called color mutants, 16 the nature of background mutations, 17 and sources of assay variability 18 have also been published. In addition, a commercial kit-based version of the assay—together with technical support, a step-by-step protocol, and accessory services, such as mutational analysis by DNA sequencing and two-dimensional electrophoresis—is being offered by Leven ( ). Our overall experience with the assay is positive, but attention must be paid to preparing DNA of high quality [even small amounts of impurities (proteins, salts, organic solvents) can sometimes decrease the rescue efficiency of the shuttle vector] and avoiding contamination with unrelated plasmids that are lacZ − , lacO + , and ampicillin resistant. Plasmids of this nature (e.g., derivatives of pBluescript) are in widespread use in many laboratories, and they can easily show up as false “pUR288 mutants” after finding their way into reagents or laboratory space where the mutagenesis assay is performed.

Fig. 4 . In vivo mutagenesis assay, using pUR288. (A) Plasmid-derived shuttle vector pUR288. CRP, cAMP receptor protein ori, origin of replication lacZ, gene encoding β-Gal HindIII, restriction site used to release the plasmid from genomic DNA. (B) Principle of the assay: LacZ− mutants are positively selected on P-Gal plates. See text for additional details.

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