Sequence Requirements for Myosin Gene Expression and Regulation in Caenorhabditis Elegans

Four Caenorhabditis elegans genes encode muscle-type specific myosin heavy chain isoforms: myo-1 and myo-2 are expressed in the pharyngeal muscles; unc-54 and myo-3 are expressed in body wall muscles. We have used transformation-rescue and lacZ fusion assays to determine sequence requirements for regulated myosin gene expression during development. Multiple tissue-specific activation elements are present for all four genes. For each of the four genes, sequences upstream of the coding region are tissue-specific promoters, as shown by their ability to drive expression of a reporter gene (lacZ) in the appropriate muscle type. Each gene contains at least one additional tissue-specific regulatory element, as defined by the ability to enhance expression of a heterologous promoter in the appropriate muscle type. In rescue experiments with unc-54, two further requirements apparently independent of tissue specificity were found: sequences within the 3' non-coding region are essential for activity while an intron near the 5' end augments expression levels. The general intron stimulation is apparently independent of intron sequence, indicating a mechanistic effect of splicing. To further characterize the myosin gene promoters and to examine the types of enhancer sequences in the genome, we have initiated a screen of C. elegans genomic DNA for fragments capable of enhancing the myo-2 promoter. The properties of enhancers recovered from this screen suggest that the promoter is limited to muscle cells in its ability to respond to enhancers.     

Endogenous retroviruses and human evolution

Humans share about 99% of their genomic DNA with chimpanzees and bonobos; thus, the differences between these species are unlikely to be in gene content but could be caused by inherited changes in regulatory systems. Endogenous retroviruses (ERVs) comprise approximately 5% of the human genome. The LTRs of ERVs contain many regulatory sequences, such as promoters, enhancers, polyadenylation signals and factor-binding sites. Thus, they can influence the expression of nearby human genes. All known human-specific LTRs belong to the HERV-K (human ERV) family, the most active family in the human genome. It is likely that some of these ERVs could have integrated into regulatory regions of the human genome, and therefore could have had an impact on the expression of adjacent genes, which have consequently contributed to human evolution. This review discusses possible functional consequences of ERV integration in active coding regions.     

Genome-Wide Characterization of Endogenous Retroviruses in the Bat Myotis lucifugus Reveals Recent and Diverse Infections

Bats are increasingly recognized as reservoir species for a variety of zoonotic viruses that pose severe threats to human health. While many RNA viruses have been identified in bats, little is known about bat retroviruses. Endogenous retroviruses (ERVs) represent genomic fossils of past retroviral infections and, thus, can inform us on the diversity and history of retroviruses that have infected a species lineage. Here, we took advantage of the availability of a high-quality genome assembly for the little brown bat, Myotis lucifugus, to systematically identify and analyze ERVs in this species. We mined an initial set of 362 potentially complete proviruses from the three main classes of ERVs, which were further resolved into 13 major families and 86 subfamilies by phylogenetic analysis. Consensus or representative sequences for each of the 86 subfamilies were then merged to the Repbase collection of known ERV/long terminal repeat (LTR) elements to annotate the retroviral complement of the bat genome. The results show that nearly 5% of the genome assembly is occupied by ERV-derived sequences, a quantity comparable to findings for other eutherian mammals. About one-fourth of these sequences belong to subfamilies newly identified in this study. Using two independent methods, intraelement LTR divergence and analysis of orthologous loci in two other bat species, we found that the vast majority of the potentially complete proviruses identified in M. lucifugus were integrated in the last ∼25 million years. All three major ERV classes include recently integrated proviruses, suggesting that a wide diversity of retroviruses is still circulating in Myotis bats.     

Glucose 6-phosphate dehydrogenase expression is less prone to variegation when driven by its own promoter

The ability to transfer permanently genes into mammalian cells makes retroviruses suitable vectors for the ultimate purpose of treating inherited genetic disease. However, expression of the retrovirally transferred genes is variable (position effect and expression variegation) because retroviruses are highly susceptible to the influence of the host genome sequences which flank the integration site. We have investigated this phenomenon with respect to the human housekeeping enzyme, glucose 6-phosphate dehydrogenase (hG6PD). We have constructed retroviral vectors in which the hG6PD cDNA is driven by either of two conventional retroviral promoters and enhancers from the Moloney Murine Leukemia Virus (MMLV) and the Myeloproliferative Sarcoma Virus (MPSV) long terminal repeats (LTR) or by the hG6PD own promoter replacing most of enhancer and promoter LTR (GRU5). We have compared the activity of retrovirally transferred hG6PD driven by these promoters after retroviral integration in bulk cultures and in individual clones of murine fibroblasts. The level of hG6PD expressed by the hG6PD promoter of GRU5-G6PD was significantly lower than that expressed by conventional retroviral vectors. However, analysis of the single copy clones showed less variation of expression with GRU5-G6PD (coefficient of variation, CV, 35.5%) than with conventional vectors (CV, 58.9%). Thus we have several vectors competent for reliable transfer and expression of hG6PD. The hG6PD promoter provides reproducible expression of hG6PD and limits the variability of expression. This decreased variability is important in order to help ensuring a consistent level of delivery of the needed gene product in future therapeutic protocols.