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Selection, recombination and demographic history in Drosophila miranda.

Selection, recombination, and the demographic history of a species can all have profound effects on genomewide patterns of variability. To assess the impact of these forces in the genome of Drosophila miranda, we examine polymorphism and divergence patterns at 62 loci scattered across the genome. In accordance with recent findings in D. melanogaster, we find that noncoding DNA generally evolves more slowly than synonymous sites, that the distribution of polymorphism frequencies in noncoding DNA is significantly skewed toward rare variants relative to synonymous sites, and that long introns evolve significantly slower than short introns or synonymous sites. These observations suggest that most noncoding DNA is functionally constrained and evolving under purifying selection. However, in contrast to findings in the D. melanogaster species group, we find little evidence of adaptive evolution acting on either coding or noncoding sequences in D. miranda. Levels of linkage disequilibrium (LD) in D. miranda are comparable to those observed in D. melanogaster, but vary considerably among chromosomes. These patterns suggest a significantly lower rate of recombination on autosomes, possibly due to the presence of polymorphic autosomal inversions and/or differences in chromosome sizes. All chromosomes show significant departures from the standard neutral model, including too much heterogeneity in synonymous site polymorphism relative to divergence among loci and a general excess of rare synonymous polymorphisms. These departures from neutral equilibrium expectations are discussed in the context of nonequilibrium models of demography and selection.

Animals↗

[Aldolase isozyme gene: on the structure and the tissue-specific expression of a muscle type aldolase gene].

A muscle-type aldolase gene known to be a tumor marker enzyme was analyzed. It was found that three different molecular species of the mRNA exist and are expressed in a tissue-specific manner. These mRNAs have identical coding and 3' noncoding sequences and differ only at the 5' end of the sequence. Genomic DNA analysis indicated that a single aldolase gene for one muscle type specifies three different mRNAs by organizing as a leader sequence a region corresponding to each distinct 5' end of the mRNA followed by a shared common structural gene in the genome.

Animals↗

Protein coding palindromes are a unique but recurrent feature in Rickettsia.

Rickettsia are unique in inserting in-frame a number of palindromic sequences within protein coding regions. In this study, we extensively analyzed repeated sequences in the genome of Rickettsia conorii and examined their locations in regard to coding versus noncoding regions. We identified 656 interspersed repeated sequences classified into 10 distinct families. Of the 10 families, three palindromic sequence families showed clear cases of insertions into open reading frames (ORFs). The location of those in-frame insertions appears to be always compatible with the encoded protein three-dimensional (3-D) fold and function. We provide evidence for a progressive loss of the palindromic property over time after the insertions. This comprehensive study of Rickettsia repeats confirms and extends our previous observations and further indicates a significant role of selfish DNAs in the creation and modification of proteins.

Amino Acid Sequence↗

Nucleotide sequence of the last exon of the gene for human cytochrome c oxidase subunit VIb and its flanking regions.

A human genomic clone encompassing the last exon of the gene for cytochrome c oxidase subunit VIb and a human genomic clone containing the most distal end of this gene were characterized. The last exon of the gene codes for the 17 C-terminal amino acid residues of the subunit and the 3' noncoding region. Downstream from the gene we found a single base difference between the DNA sequences of the two genomic clones. An inverted Alu dimer repeat was identified further downstream.

Amino Acid Sequence↗

Identification of two novel 5' noncoding exons in human MNB/DYRK gene and alternatively spliced transcripts.

Previously, the comparison of human MNB/DYRK cDNA sequences with publicly available genomic DNA sequence made a conclusion that MNB/DYRK gene consists of 11 exons spanning 100 kb. Here we found a novel human MNB/DYRK cDNA clone that has the 5' end different from any of those reported MNB/DYRK cDNAs. The 5' end of this cDNA resides in a CpG island associated with the NotI linking clone LL390 that is located more than 50 kb upstream of the exon that contains an initiation codon ATG. Northern blot analysis indicated that almost all the MNB/DYRK transcripts start from the newly identified 5'-noncoding exon. PCR amplification of cDNAs from various human tissues indicated that some mRNAs contain an additional noncoding exon of 76 bp next to the 5'-end exon and there are at least three types of MNB/DYRK mRNAs containing alternatively spliced 5' exons including two novel exons. We now conclude that human MNB/DYRK gene consists of 13 exons (not 11 exons) spanning about 150 kb (not 100 kb).

Alternative Splicing↗

Construction and mutagenesis of an artificial bicistronic tick-borne encephalitis virus genome reveals an essential function of the second transmembrane region of protein e in flavivirus assembly.

Flaviviruses have a monopartite positive-stranded RNA genome, which serves as the sole mRNA for protein translation. Cap-dependent translation produces a polyprotein precursor that is co- and posttranslationally processed by proteases to yield the final protein products. In this study, using tick-borne encephalitis virus (TBEV), we constructed an artificial bicistronic flavivirus genome (TBEV-bc) in which the capsid protein and the nonstructural proteins were still encoded in the cap cistron but the coding region for the surface proteins prM and E was moved to a separate translation unit under the control of an internal ribosome entry site element inserted into the 3' noncoding region. Mutant TBEV-bc was shown to produce particles that packaged the bicistronic RNA genome and were infectious for BHK-21 cells and mice. Compared to wild-type controls, however, TBEV-bc was less efficient in both RNA replication and infectious particle formation. We took advantage of the separate expression of the E protein in this system to investigate the role in viral assembly of the second transmembrane region of protein E (E-TM2), a second copy of which was retained in the cap cistron to fulfill its other role as an internal signal sequence in the polyprotein. Deletion analysis and replacement of the entire TBEV E-TM2 region with its counterpart from another flavivirus revealed that this element, apart from its role as a signal sequence, is important for virion formation.

Amino Acid Sequence↗

Evolutionary parameters of the transcribed mammalian genome: an analysis of 2,820 orthologous rodent and human sequences.

We have rigorously defined 2,820 orthologous mRNA and protein sequence pairs from rats, mice, and humans. Evolutionary rate analyses indicate that mammalian genes are evolving 17-30% more slowly than previous textbook values. Data are presented on the average properties of mRNA and protein sequences, on variations in sequence conservation in coding and noncoding regions, and on the absolute and relative frequencies of repetitive elements and splice sites in untranslated regions of mRNAs. Our data set contains 1,880 unique human/rodent sequence pairs that represent about 2-4% of all mammalian genes. Of the 1,880 human orthologs, 70% are present on a new gene map of the human genome, thus providing a valuable resource for cross-referencing human and rodent genomes. In addition to comparative mapping, these results have practical applications in the interpretation of noncoding sequence conservation between syntenic regions of human and mouse genomic sequence, and in the design and calibration of gene expression arrays.

Animals↗

The 5' noncoding region of the type 2 poliovirus vaccine strain contains determinants of attenuation and temperature sensitivity.

Intratypic recombinants of P2/Sabin and P2/117, a neurovirulent vaccine revertant, have been generated in vitro using infectious cDNA clones and used to demonstrate that strong determinants of the attenuation and temperature-sensitive phenotypes of P2/Sabin reside in the 5' 492 nucleotides. In this region of the genome the viruses differ only at nucleotides 437 and 481. The ts phenotype associated with the 5' noncoding region is expressed at different temperatures in different cell lines, suggesting an involvement of cellular factors which may be species specific. Suppression of both the ts and attenuation phenotypes correlates with an A-G mutation at nucleotide 481, although other changes are also involved.

Animals↗

Molecular cloning of the rat thyroxine-binding globulin gene and analysis of its promoter activity.

We cloned the rat T4-binding globulin (rTBG) gene, characterized its organization, and studied its promoter activity and regulation. A genomic DNA library was constructed and screened using an rTBG complementary DNA (cDNA) as a probe. An 8.6-kilobase pair (kbp) clone was partially sequenced and compared with the sequence of the previously cloned cDNA. It helped complete the cDNA sequence and identify the first noncoding exon (exon 0). The transcription start site was identified using an RNase protection assay. The rTBG genomic clone contained 1.2 kbp 5'-flanking and 1.7 kbp 3'-flanking regions. The sizes of exons and introns of the rTBG gene are similar to those of the human TBG gene, belonging to the serine protease inhibitor family. The 5'-flanking region contains a TATA box, a CAAT box, and a consensus sequence for the hepatocyte nuclear factor 1-binding site. We tested the promoter activity of the 1.2-kbp 5'-flanking region using a luciferase reporter plasmid. When transfected into a hepatocyte-derived cell line (HepG2), the plasmid construct containing the fragment -1227 to +11 (transcription start site, +1) showed a 9-fold increase in luciferase activity compared with that of a promoterless luciferase vector. No promoter activity was detected in a nonhepatocyte-derived cell line (COS1). Serial 5'-deletion revealed that the construct containing the fragment -180 to +11 had 40% of the maximal promoter-induced luciferase activity. And that containing the fragment -53 to +11 showed no significant increase of luciferase activity. These results suggest that the region -180 to -53, containing hepatocyte nuclear factor 1-binding site, is essential to the liver-specific expression. We previously reported down-regulation of rTBG messenger RNA by T3 in vivo. The present study failed to show T3's effect on the promoter activity of the 1.2-kbp 5'-flanking region of the rTBG gene.

Animals↗

Distinguishing regulatory DNA from neutral sites.

We explore several computational approaches to analyzing interspecies genomic sequence alignments, aiming to distinguish regulatory regions from neutrally evolving DNA. Human-mouse genomic alignments were collected for three sets of human regions: (1) experimentally defined gene regulatory regions, (2) well-characterized exons (coding sequences, as a positive control), and (3) interspersed repeats thought to have inserted before the human-mouse split (a good model for neutrally evolving DNA). Models that potentially could distinguish functional noncoding sequences from neutral DNA were evaluated on these three data sets, as well as bulk genome alignments. Our analyses show that discrimination based on frequencies of individual nucleotide pairs or gaps (i.e., of possible alignment columns) is only partially successful. In contrast, scoring procedures that include the alignment context, based on frequencies of short runs of alignment columns, dramatically improve separation between regulatory and neutral features. Such scoring functions should aid in the identification of putative regulatory regions throughout the human genome.

Animals↗

Specific detection of hepatitis C virus minus strand RNA in hematopoietic cells.

The presence of hepatitis C virus (HCV) negative strand RNA in extrahepatic compartments based on PCR detection assays has been suggested in many reports with a very heterologous detection rate (from 0 to 100%). In this study, we have analyzed the presence of HCV negative strand in hepatic (liver biopsies, n = 20) and extrahepatic (sera, n = 32; PBMC, n = 26 and fresh bone marrow cells, n = 8) compartments from infected patients with three different reverse transcriptase (RT)-PCR-based assays using primers located in the 5' noncoding region, with or without a tag selected to display different viral loads (10(5)-3 x 10(7) genomic equivalent/ml or gram) and viral genotypes (n = 5). Using synthetic as well as biological templates, we could document extensive artifactual detection of negative strand RNA, due to self priming and mispriming events, even either 5' noncoding region primer pair was used, whereas both artifacts were dramatically reduced (mispriming) or eliminated (selfpriming) using CAP-based RT-PCR assay. Mispriming artifacts were directly correlated to the titer of positive strand RNA present in the sample. Using the CAP-PCR assay, the presence of HCV negative strand RNA was found in 75% of livers (16:20) and only 8% of PBMC, independent of the genotype involved, but could not be documented in sera (0:32) and fresh bone marrow cells (0:6). These findings suggest that caution regarding the type of RT-PCR assay used and the level of HCV positive strand RNA present in the biological sample analyzed has to be taken to avoid false identification of viral reservoirs. The findings suggest that hematopoietic peripheral cells can support HCV replication, although in a very limited number of carriers.

Artifacts↗

The La antigen binds 5' noncoding region of the hepatitis C virus RNA in the context of the initiator AUG codon and stimulates internal ribosome entry site-mediated translation.

Translation initiation of the hepatitis C virus (HCV) RNA genome occurs through an internal ribosome entry site in a cap-independent manner. Here, we have examined the interaction between La antigen and the HCV 5' noncoding region (5'NCR). In this analysis, competitor RNAs derived from HCV 5'NCR carrying deletions and a point mutation were used to identify the site(s) of La antigen binding during UV cross-linking assay. These studies suggest that La antigen recognizes the intact HCV 5'NCR structure. Further, these interactions occurred in the context of the initiator AUG. The latter view is supported by an analysis in which mutants of the HCV 5'NCR RNA with deletion or substitution in the initiator AUG codon failed to compete for La antigen binding to the wild-type 5'NCR. The evidence for the interaction between liver cell-derived La antigen and the HCV 5'NCR is provided by immunoprecipitation of a UV cross-linked species from the S100 fraction of Huh7 cell lysates. The functional relevance of this interaction was demonstrated by the stimulation of the HCV internal ribosome entry site-mediated translation in the presence of La protein. These results suggest an important functional role of La protein in the regulation of internal initiation of translation of the HCV RNA genome.

Autoantigens↗

A bird's-eye view of the C-value enigma: genome size, cell size, and metabolic rate in the class aves.

For half a century, variation in genome size (C-value) has been an unresolved puzzle in evolutionary biology. While the initial "C-value paradox" was solved with the discovery of noncoding DNA, a much more complex "C-value enigma" remains. The present study focuses on one aspect of this puzzle, namely the small genome sizes of birds. Significant negative correlations are reported between resting metabolic rate and both C-value and erythrocyte size. Cell size is positively correlated with both nucleus size and C-value in birds, as in other vertebrates. These findings shed light on the constraints acting on genome size in birds and illustrate the importance of interactions among various levels of the biological hierarchy, ranging from the subchromosomal to the ecological. Following from a discussion of the mechanistic bases of the correlations reported and the processes by which birds achieved and/or maintain small genomes, a pluralistic approach to the C-value enigma is recommended.

Animals↗

Full-length cDNAs: more than just reaching the ends.

The development of functional genomic resources is essential to understand and utilize information generated from genome sequencing projects. Central to the development of this technology is the creation of high-quality cDNA resources and improved technologies for analyzing coding and noncoding mRNA sequences. The isolation and mapping of cDNAs is an entrée to characterizing the information that is of significant biological relevance in the genome of an organism. However, a bottleneck is often encountered when attempting to bring to full-length (or at least full-coding) a number of incomplete cDNAs in parallel, since this involves the nonsystematic, time consuming, and labor-intensive iterative screening of a number of cDNA libraries of variable quality and/or directed strategies to process individual clones (e.g., 5' rapid amplification of cDNA ends). Here, we review the current state of the art in cDNA library generation, as well as present an analysis of the different steps involved in cDNA library generation.

Automation↗

A novel class of developmentally regulated noncoding RNAs in Leishmania.

Leishmania is a protozoan parasite that causes serious morbidity and mortality in humans worldwide. The ability of these parasites to survive within the phagolysosomes of mammalian macrophages is dependent on the developmental regulation of a variety of genes. Identifying genomic sequences that are preferentially expressed during the parasite's intracellular growth would provide new insights about the mechanisms controlling stage-specific gene regulation for intracellular development of the parasite. Using a genomic library that differentially hybridized to probes made from total RNA from Leishmania infantum amastigote or promastigote life cycle stages, we identified a new class of noncoding RNAs (ncRNAs) ranging from approximately 300 to 600 nucleotides in size that are expressed specifically in the intracellular amastigote stage. These ncRNAs are transcribed by RNA polymerase II from genomic clusters of tandem head-to-tail repeats, which are mainly located within subtelomeric regions. Remarkably, both the sense and antisense orientations of these ncRNAs are transcribed and are processed by trans splicing and polyadenylation. The levels of antisense transcripts are at least 10-fold lower than those of the sense transcripts and are tightly regulated. The sense and antisense ncRNAs are cytosolic as shown by fluorescence in situ hybridization studies and cosediment with a small ribonucleoprotein complex. Amastigote-specific regulation of these ncRNAs possibly occurs at the level of RNA stability. Interestingly, overexpression of these ncRNAs in promastigotes, as part of an episomal expression vector, failed to produce any transcript, which further highlights the instability of these RNAs in the promastigote stage. This is the first report describing developmentally regulated ncRNAs in protozoan parasites.

Animals↗

Polymorphism of unique noncoding DNA sequences in wild and laboratory mice.

Two DNA probes, D17Tu1 and D17Tu2, were isolated from a genomic DNA library containing only two mouse chromosomes, one of which is chromosome 17, carrying the major histocompatibility complex (H-2), as well as the t complex genes. The D17Tu1 probe was mapped to the centromeric region of chromosome 17 and the D17Tu2 probe to the S region of the H-2 complex. Neither of the two probes appeared to detect any genes, but both contained unique, nonrepetitive sequences. Typing of DNA obtained from a large panel of mice revealed the presence of four D17Tu1 patterns in inbred mouse strains, one very common, one less common, and two present in one strain each. The two common patterns could not be detected in appreciable frequencies in the European wild mice tested (one of the two patterns was, however, found in Australian wild mice). Conversely, the patterns found frequently in European wild mice are absent in the laboratory mice. We therefore conclude that wild mice from the sampled regions of Europe could not have provided the ancestral stocks from which inbred strains were derived. Only one D17Tu1 pattern was found in all the populations of Mus musculus tested, while eight patterns were found in Mus domesticus, with virtually all the populations being polymorphic. We suggest that this difference reflects different modes in which the two species colonized Europe. The distribution of the D17Tu2 patterns in inbred strains correlates with the distribution of H-2 haplotypes.

Animals↗

Structural and functional characterization of the rat neurotrophin-4 gene.

Neurotrophin-4 (NT-4) is a member of the neurotrophin family of growth factors. To study the molecular mechanisms that govern NT-4 expression, we have cloned and characterized the rat genome region encoding NT-4. The rat NT-4 gene consists of three exons: two 5'-flanking noncoding exons and a coding exon. NT-4 mRNA transcription is controlled by two promoters flanking the noncoding exons. Alternative splicing of the second intron results in a NT-4 mRNA with a different open reading frame, encoding a shorter protein lacking pre-NT-4 sequence. A rat NT-4 gene fragment, containing all exons and introns in addition to 1.4 kb of the upstream genomic sequence, has been introduced into mice. This transgene enables partial recapitulation of the expression pattern of NT-4 mRNA and confers activity-dependent expression of the NT-4 mRNA in muscle.

Animals↗

The role of context-dependent mutations in generating compositional and codon usage bias in grass chloroplast DNA.

The influence of local base composition on mutations in chloroplast DNA (cpDNA) is studied in detail and the resulting, empirically derived, mutation dynamics are used to analyze both base composition and codon usage bias. A 4 x 4 substitution matrix is generated for each of the 16 possible flanking base combinations (contexts) using 17,253 noncoding sites, 1309 of which are variable, from an alignment of three complete grass chloroplast genome sequences. It is shown that substitution bias at these sites is correlated with flanking base composition and that the A+T content of these flanking sites as well as the number of flanking pyrimidines on the same strand appears to have general influences on substitution properties. The context-dependent equilibrium base frequencies predicted from these matrices are then applied to two analyses. The first examines whether or not context dependency of mutations is sufficient to generate average compositional differences between noncoding cpDNA and silent sites of coding sequences. It is found that these two classes of sites exist, on average, in very different contexts and that the observed mutation dynamics are expected to generate significant differences in overall composition bias that are similar to the differences observed in cpDNA. Context dependency, however, cannot account for all of the observed differences: although silent sites in coding regions appear to be at the equilibrium predicted, noncoding cpDNA has a significantly lower A+T content than expected from its own substitution dynamics, possibly due to the influence of indels. The second study examines the codon usage of low-expression chloroplast genes. When context is accounted for, codon usage is very similar to what is predicted by the substitution dynamics of noncoding cpDNA. However, certain codon groups show significant deviation when followed by a purine in a manner suggesting some form of weak selection other than translation efficiency. Overall, the findings indicate that a full understanding of mutational dynamics is critical to understanding the role selection plays in generating composition bias and sequence structure.

Base Composition↗