Germ line transposition of the copia retrotransposon in Drosophila melanogaster is restricted to males by tissue-specific control of copia RNA levels

Germ line transposition rates of the retrotransposon copia were directly measured in males and females of an inbred Drosophila melanogaster line, 2b3, which is highly polymorphic for copia insertion sites. The elevated germ line transposition rate of copia in this line (3–8?×?10^?3 per generation per element) is confined to males, with transposition in females being undetectable under the conditions of the experiment but at most 50-fold lower than the rate for males. To determine the molecular basis of this effect, copia RNA levels were measured in whole bodies and germ lines of male and female flies of both the unstable 2b3 line and a stable line, Oregon RC-iso, which shows normal rates of copia transposition. Both male and female 2b3 flies contain much more copia RNA than flies of the stable line. However, 2b3 male germinal tissues contain much higher levels of copia RNA than the equivalent female tissues. The highest copia expression is detected in maturing primary spermatocytes. Our data show that high rates of germ line copia transposition are restricted to males by tissue-specific control of RNA levels and suggest that transposition of copia only occurs in fly tissues containing more than a relatively high threshold level of copia RNA.     

Age dependence of the level of copia retrotransposon expression in the testes of Drosophila melanogaster

Expression of the lacZ reporter gene controlled by various deletion derivatives of the regulatory region of the copia retrotransposon was studied in the testes of adult transgenic males of the Drosophila melanogaster y1W67c23(2) strain at the age of 3, 6-9, 12-15, 18-21, and 24-27 days. When the construct contained the full-length regulatory region, which included the 5'-long terminal repeat (LTR) and the 5'-untranslated region (UTR), expression was the lowest in males aged 12-15 days and the highest in males aged 3 or 24-27 days. A similar V-shaped age dependence was previously observed for the copia transposition rate and RNA content in a strain with a high rate of copia transposition. Thus, the V-shaped age dependence of expression, which is unusual for Drosophila, proved to be characteristic of copia regardless of its transposition rate. Deletion of the 5' or 3' end of the LTR, but not of the UTR, changed the age dependence of the level of reporter gene expression. In this case, expression was the highest in 3-day-old males and gradually decreased with age, as typical for many Drosophila genes. It was assumed that the 5'- and 3'-terminal regions of the copia LTR contain regulatory elements responsible for the V-shaped age dependence of expression, while the expression level depends to a greater extent on the regulatory elements of UTR.     

Tissue specificity of epithelial keratins: differential expression of mRNAs from two multigene families.

Human epithelial cells cultured from stratified and simple squamous tissues all produce keratins of 40,000 to 58,000 daltons, but within this range the number and sizes vary with different epithelial cells. We have shown that this tissue-specific variation in the keratins is not due to posttranslational modification or processing, but rather to the differential expression of a family of heterogeneous but closely related mRNAs. All of these epithelial keratin mRNAs can be further grouped into two distinct subfamilies by their ability to hybridize with either of two cloned epidermal keratin cDNAs. All of the keratin mRNAs hybridize to one or the other, but not both, of the two cloned cDNAs. However, the mRNAs within each group hybridize with varying degrees of stringency, indicating that they are of similar but not identical sequence. Both types of keratin mRNAs are always expressed in every epithelial cell line studied, suggesting that filament assembly is dependent on the presence of both types of keratins. Within each of these two groups, the slight sequence differences in each class may reflect subtle tissue-specific variations in the structural and functional requirements of the epithelial cytoskeleton.     

RNA sequencing reveals two major classes of gene expression levels in metazoan cells

The expression level of a gene is often used as a proxy for determining whether the protein or RNA product is functional in a cell or tissue. Therefore, it is of fundamental importance to understand the global distribution of gene expression levels, and to be able to interpret it mechanistically and functionally. Here we use RNA sequencing (RNA‐seq) of mouse Th2 cells, coupled with a range of other techniques, to show that all genes can be separated, based on their expression abundance, into two distinct groups: one group comprised of lowly expressed and putatively non‐functional mRNAs, and the other of highly expressed mRNAs with active chromatin marks at their promoters. These observations are confirmed in many other microarray and RNA‐seq data sets of metazoan cell types.     

Multiplex relative RT-PCR method for verification of differential gene expression

Differential display, suppression subtractive hybridization and other techniques for identification of differentially expressed genes produce fragments of cDNA from mRNAs whose differences in abundance must be verified. This report describes a relative multiplex RT-PCR assay that facilitates the analysis of large numbers of samples for differences in mRNA abundance without the use of radioactivity or blotting. The species of interest is co-amplified with 18S rRNA over a range of cycles followed by electrophoresis through ethidium bromide-agarose gels. Intensities of the bands of interest, normalized for 18S band intensities, are plotted as a function of cycle number. Regression equations fitted to the curves are used to calculate the number of cycles necessary for each sample's normalized signal to reach a threshold intensity. Differences between samples in the number of cycles required to reach that threshold reflect differences in the original abundances of those species. A comparison with results previously obtained using northern blots showed that relative differences as small as 20% and as large as an order of magnitude are accurately detected. The simplicity of the assay allows its routine application in both research and teaching laboratories.     

Chicken nonmuscle myosin heavy chains: differential expression of two mRNAs and evidence for two different polypeptides

Two different mRNAs encoding two different nonmuscle myosin heavy chains (MHCs) of approximately 200 kD have been identified in chicken nonmuscle cells, in agreement with the results of Katsuragawa et al. (Katsuragawa, Y., M. Yanagisawa, A. Inoue, and T. Masaki. 1989. Eur. J. Biochem. 184:611-616). In this paper, we quantitate the content of mRNA encoding the two MHCs in a number of different tissues using RNA blot analysis with two specific oligonucleotide probes. Our results show that the relative content of mRNA encoding MHC-A and MHC-B differs in a tissue-dependent manner. Thus the ratio of mRNA encoding MHC-A versus MHC-B varies from greater than 9:1 in spleen and intestinal epithelial cells, to 6:4 in kidney and 2:8 in brain. The effect of serum on MHC mRNA expression was studied in serum-starved cultures of chick embryo fibroblasts. Serum stimulation results in a threefold increase in the mRNA encoding MHC-A and a threefold decrease in mRNA encoding MHC-B. Using SDS polyacrylamide gels, we have separated two nonmuscle MHC isoforms (198 and 196 kD) that can be distinguished from each other by two-dimensional peptide mapping of chymotryptic digests. We provide preliminary evidence that the MHC-A mRNA encodes the 196-kD polypeptide and that the MHC-B mRNA encodes the 198-kD polypeptide.     

Class I major histocompatibility gene products of the brown Norway rat display two major antigenic regions

Sixteen Lewis anti-Brown Norway monoclonal antibodies and sera from alloimmunized Lewis rats were used to study the topographic relationships of antigenic determinants on class I major histocompatibility gene products. Only two independent antigenic regions were identified in competition binding assays. The first region is composed of a set of overlapping epitopes that are conserved in class I major histocompatibility products of mice and humans, as well as rats. In contrast, the second antigenic region appears in a restricted number of inbred rat strains and is not detected in other species. The data provide serologic confirmation, at the monoclonal and serum alloantibody level, of conserved polymorphisms in the major histocompatibility gene products of different species, a finding that is consistent with the amino acid sequences of these molecules.     

Regulation of PCNA and CAF-1 expression by the two tuberous sclerosis gene products

Tuberous sclerosis is an autosomal dominant tumor suppressor gene syndrome affecting about 1 in 6000 individuals. Two genes have been shown to be responsible for this disease: TSC1, encoding hamartin and TSC, encoding tuberin. A variety of tumors characteristically occur in different organs of tuberous sclerosis patients and are believed to result from defects in cell cycle/cell size control. In this study, we performed two-dimensional gel electrophoresis with subsequent mass spectrometrical identification of protein spots after overexpression of TSC1 or TSC2. We found expression of PCNA and the p48 subunit of CAF-1 to be regulated by two tuberous sclerosis gene products. CAF-1 and PCNA interact as major regulators of chromatin assembly during DNA repair. We suggest that deregulation of the control of chromatin assembly might contribute to development of tumors in tuberous sclerosis patients and provide important new insights into the molecular development, especially since deregulation of chromatin assembly and DNA repair results in genomic instability, a hallmark of tumor development.     

The INK4A/ARF locus and its two gene products

The INK4A/ARF locus on chromosome 9 is one of the sites mutated most frequently in human cancer. Two genes comprising overlapping reading frames encoding p16(INK4a) and p19(ARF) have been discovered at this locus and, remarkably, both play an important role in regulating cell growth, survival and senescence.     

Expression and differential intracellular localization of two major forms of human 8-oxoguanine DNA glycosylase encoded by alternatively spliced OGG1 mRNAs

We identified seven alternatively spliced forms of human 8-oxoguanine DNA glycosylase (OGG1) mRNAs, classified into two types based on their last exons (type 1 with exon 7: 1a and 1b; type 2 with exon 8: 2a to 2e). Types 1a and 2a mRNAs are major in human tissues. Seven mRNAs are expected to encode different polypeptides (OGG1-1a to 2e) that share their N terminus with the common mitochondrial targeting signal, and each possesses a unique C terminus. A 36-kDa polypeptide, corresponding to OGG1-1a recognized only by antibodies against the region containing helix-hairpin-helix-PVD motif, was copurified from the nuclear extract with an activity introducing a nick into DNA containing 8-oxoguanine. A 40-kDa polypeptide corresponding to a processed form of OGG1-2a was detected in their mitochondria using antibodies against its C terminus. Electron microscopic immunocytochemistry and subfractionation of the mitochondria revealed that OGG1-2a locates on the inner membrane of mitochondria. Deletion mutant analyses revealed that the unique C terminus of OGG1-2a and its mitochondrial targeting signal are essential for mitochondrial localization and that nuclear localization of OGG1-1a depends on the NLS at its C terminus.