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PubMed1984

A 3' co-terminal family of mRNAs from the herpes simplex virus type 1 short region: two overlapping reading frames encode unrelated polypeptide one of which has highly reiterated amino acid sequence.

We have used DNA sequencing, mRNA mapping and in vitro translation to characterise three partially overlapping genes in the genome of herpes simplex virus (HSV) type 1. These genes specify three mRNAs with distinct 5' termini but a common 3' terminus, the longest of which is immediate-early (IE) mRNA-5. The 12,000 MW (12K) IE polypeptide encoded by IEmRNA-5 is translated from an 88 codon open reading frame, leaving a 1200 base 3' non-translated region. The second mRNA (mRNA-B) is initiated within the coding sequence of IEmRNA-5, and encodes a 21K polypeptide. The 12K and 21K polypeptide coding regions do not overlap. The third mRNA (mRNA-C) is initiated within the coding region of mRNA-B, and encodes a 33K polypeptide. The reading frame for 33K has a 110 codon out-of-frame overlap with the 21K reading frame. This is the first instance of overlapping genes described for HSV. The 21K polypeptide is thought to be a DNA binding protein and is remarkable for an array of 24 tandem repeats of the sequence X/Pro/Arg (where X represents predominantly Glu, Asp, Thr, Ser or Val) in its C-terminal portion. This array, which occupies most of the region of overlap with 33K, can vary in repeat number between virus strains.

Amino Acid Sequence↗
PubMed1991

Phenotypic and genotypic characterization of locus Syn 5 in herpes simplex virus 1.

Previous papers have reported that the syncytial mutant HSV-1(13)S11 carries three segregable syn mutations and exhibits its altered phenotype in four different cell lines, i.e. HEp-2, VERO, BHK and HEL both at 34 degrees C and 39 degrees C. Those studies have shown that one of three syncytial loci, designated Syn 5, is located in the Bam HI Q fragment spanning map units 0.296-0.317 of the prototype arrangement. Recombinants obtained from marker transfer experiments with donor BamHI Q fragment, have shown that locus Syn 5 is able to induce cell-to-cell fusion in VERO, BHK and HEL but not in HEp-2 cells. In this paper we have characterized the syn mutant HSV-1(13)S11 with regard to plaque morphology, synthesis of viral polypeptides and glycoproteins, thymidine kinase activity and physical map position of locus Syn 5 on the genome. Pertinent to the syn phenotype, earlier papers claimed that two different polypeptides, thymidine kinase (TK) and glycoprotein H (gH), whose genes map in BamHI Q, may be responsible for the fusion activity. Functional studies on the TK of the syn mutant HSV-1(13)S11 indicate that this polypeptide accumulates normally in infected cells and is a fully active enzyme. The other gene product, gH, has been studied with SDS-PAGE and in radioimmunoprecipitation (RIP) experiments using specific monoclonal antibodies. The results indicate that the amount of gH accumulation in the syn mutant-infected cells is greater than its parental strain. However, new marker transfer experiments described here located locus Syn 5 in 663 base pairs between SstI and EcoRI restriction endonuclease sites at the right end of the BamHI Q fragment, where TK gene overlaps in opposite orientation with UL 24 gene. Altogether these results indicate that the Syn 5 locus segregates from the gene specifying gH, to a region encompassing portions of the TK and UL 24 genes, and that the syn mutation does not affect the expression or activity of TK.

Animals↗
PubMed1991

High-level ribosomal frameshifting directs the synthesis of IS150 gene products.

IS150 contains two tandem, out-of-phase, overlapping genes, ins150A and ins150B, which are controlled by the same promoter. These genes encode proteins of 19 and 31 kD, respectively. A third protein of 49 kD is a transframe gene product consisting of domains encoded by both genes. Specific -1 ribosomal frameshifting is responsible for the synthesis of the large protein. Expression of ins150B also involves frameshifting. The IS150 frameshifting signals operate with a remarkably high efficiency, causing about one third of the ribosomes to switch frame. All of the signals required for this process are encoded in a 83-bp segment of the element. The heptanucleotide A AAA AAG and a potential stem-loop-forming sequence mark the frameshifting site. Similar sequence elements are found in -1 frameshifting regions of bacterial and retroviral genes. A mutation within the stem-loop sequence reduces the rate of frameshifting by about 80%. Artificial transposons carrying this mutation transpose at a normal frequency, but form cointegrates at a approximately 100-fold reduced rate.

Amino Acid Sequence↗
PubMed1989

Overlapping transcription and termination of the convergent ilvA and ilvY genes of Escherichia coli.

The ilvY gene of Escherichia coli is transcribed in a direction opposite to the ilvGMEDA operon, and the translational stop codons of the ilvA and ilvY genes are only 52 base pairs (bp) apart. We have employed galK transcriptional analyses, in vitro transcription assays, and S1 nuclease mapping to show that the converging transcripts of the ilvGMEDA operon and the ilvY gene overlap. The ilvGMEDA transcript terminates at two sites in the distal amino acid coding region of the ilvY gene: a rho-independent termination site 116 bp downstream of the ilvA translational stop codon, ilvA t, and a 62-bp-long rho-dependent termination site, ilvA t', beginning 70 bp beyond the ilvA t site. This tt' termination pattern at the distal end of the ilvGMEDA operon is similar to that of the trp operon of E. coli and the leu operon of Salmonella typhimurium. Termination of ilvY transcription occurs over a broad stretch of DNA several hundred base pairs downstream of the ilvY translational stop codon in the middle of the amino acid coding region of the ilvA gene. These experiments demonstrate that the converging transcripts from the ilvA and ilvY genes terminate in the coding region of the opposing gene.

Amino Acids, Branched-Chain↗
PubMed1993

Varicella-zoster virus DNA polymerase and major DNA-binding protein genes have overlapping divergent promoters.

A detailed analysis of the transcriptionally divergent promoters of varicella-zoster virus (VZV) open reading frames (ORFs) 28 and 29, encoding the DNA polymerase and major DNA-binding proteins, respectively, was performed. We found that the 221-bp ORF 28-29 intergenic domain contains overlapping divergent promoters; these promoters have TATA boxes and cap sites arranged closely back-to-back, have highly concordant patterns of responsiveness to transactivation by VZV ORFs 4 and/or 62, and could not be separated without abolishing the effects that VZV trans activators imparted to them. Mutation of the ORF 28 TATA box rendered this promoter unresponsive to ORF 62 and the combination of ORFs 4 and 62 without altering ORF 29 promoter activity. Mutations of all potential ORF 29 TATA boxes collectively failed to abolish this promoter's responsiveness to either ORF 4 or ORF 62, suggesting a mechanism of gene regulation for ORF 29 that differs from that of ORF 28. These findings are concordant with the observation that both genes are expressed in productive infection, but only ORF 29 expression has been identified in latency.

Animals↗
PubMed1997

Expression of the thyroid hormone receptor gene, erbAalpha, in B lymphocytes: alternative mRNA processing is independent of differentiation but correlates with antisense RNA levels.

The erbAalpha gene encodes two alpha-thyroid hormone receptor isoforms, TRalpha1 and TRalpha2, which arise from alternatively processed mRNAs, erbAalpha1 (alpha1) and erb alpha2 (alpha2). The splicing and alternative polyadenylation patterns of these mRNAs resemble that of mRNAs encoding different forms of immunoglobulin heavy chains, which are regulated at the level of alternative processing during B cell differentiation. This study examines the levels of erbAalpha mRNA in eight B cell lines representing four stages of differentiation in order to determine whether regulation of the alternatively processed alpha1 and alpha2 mRNAs parallels the processing of immunoglobulin heavy chain mRNAs. Results show that the pattern of alpha1 and alpha2 mRNA expression is clearly different from that observed for immunoglobulin heavy chain mRNAs. B cell lines display characteristic ratios of alpha1/alpha2 mRNA at distinct stages of differentiation. Furthermore, expression of an overlapping gene, Rev-ErbAalpha (RevErb), was found to correlate strongly with an increase in the ratio of alpha1/alpha2 mRNA. These results suggest that alternative processing of erbAalpha mRNAs is regulated by a mechanism which is distinct from that regulating immunoglobulin mRNA. The correlation between RevErb and erbAalpha mRNA is consistent with negative regulation of alpha2 via antisense interactions with the complementary RevErb mRNA.

Alternative Splicing↗
PubMed1989

Synthesis and encapsidation of duck hepatitis B virus reverse transcriptase do not require formation of core-polymerase fusion proteins.

The expression strategy of the duck hepatitis B virus (DHBV) P gene, which is assumed to encode the viral reverse transcriptase, was investigated by mutational analysis. This study showed that P gene expression starts in the region where the P gene overlaps the viral core gene. However, in contrast to retroviral reverse transcriptases, which are expressed via gag-pol fusion protein intermediates, the DHBV P gene product was found to be synthesized starting at a P gene ATG codon. The resulting protein can complement polymerase-negative mutants in trans and can reverse transcribe viral pregenomic RNA that does not encode an active polymerase. These findings raise the question of how reverse transcription of cellular RNAs can be avoided in infected cells.

Animals↗
PubMed1986

Nucleotide sequence of the woodchuck hepatitis virus surface antigen mRNAs and the variability of three overlapping viral genes.

A cDNA library was constructed from the liver of a woodchuck chronically infected with woodchuck hepatitis virus (WHV). A clone, pWS23, encompassing the entire surface and X genes of WHV was isolated. Comparison of the complete nucleotide (nt) sequence of pWS23 with those of genomic DNAs from two different WHV isolates showed that it contained a nearly full-length copy of the major mRNA encoding the viral surface antigen (S mRNA). It was colinear with the WHV genome over 1858 nt and terminated 22 nt downstream from the variant polyadenylation signal within the core gene. Evidence for heterogeneity of the 5'-terminal region of the S mRNA came from direct sequencing of the 5' extremities of 20 cDNA inserts, similar to that of pWS23, isolated from a second cDNA library of the same woodchuck liver. In agreement with previous mapping studies of hepadnaviruses, two main initiation regions of S mRNA were localized 27-30 nt upstream and 22-49 nt downstream from the pre-S2 initiation codon. Further analysis of the amino acid sequences of the surface, polymerase and X genes of WHV showed a high conservation among three WHV isolates and a similar distribution of conserved and variable regions in woodchuck and human hepatitis B viruses.

Amino Acid Sequence↗
PubMed1991

CpG islands, genes and isochores in the genomes of vertebrates.

We have shown that human genes associated with CpG islands increase in number as they increase in % of guanine + cytosine (GC) levels, and that most genes associated with CpG islands are located in the GC-richest compartment of the human genome. This is an independent confirmation of the concentration gradient of CpG islands (detected as HpaII tiny fragments, or HTF) which was demonstrated in the genome of warm-blooded vertebrates [Aïssani and Bernardi, Gene 106 (1991) 173-183]. We then reassessed the location of CpG islands using the data currently available and confirmed that CpG islands are most frequently located in the 5'-flanking sequences of genes and that they overlap genes to variable extents. We have shown that such extents increase with the increasing GC levels of genes, the GC-richest genes being completely included in CpG islands. Under such circumstances, we have investigated the properties of the 'extragenic' CpG islands located in the 5'-flanking segments of homologous genes from both warm- and cold-blooded vertebrates. We have confirmed that, in cold-blooded vertebrates, CpG islands are often absent; when present, they have lower GC and CpG levels; the latter attain, however, statistically expected values. Finally, we have shown that CpG doublets increase with the increasing GC of exons, introns and intergenic sequences (including 'extragenic' CpG islands) in the genomes from both warm- and cold-blooded vertebrates. The correlations found are the same for both classes of vertebrates, and are similar for exons, introns and intergenic sequences (including 'extragenic' CpG islands). The findings just outlined indicate that the origin and evolution of CpG islands in the vertebrate genome are associated with compositional transitions (GC increases) in genes and isochores.

Animals↗
PubMed2000

Criteria for gene identification and features of genome organization: analysis of 6.5 Mb of DNA sequence from human chromosome 21.

To establish criteria for and the limitations of novel gene identification, to identify novel genes of potential relevance to Down Syndrome and to investigate features of genome organization, 6. 550kb. In total, 41 novel gene models were predicted, and for a subset of these, RT-PCR experiments helped to verify and refine the models, and were used to assess expression in early development and in adult brain regions of potential relevance to Down syndrome. Results suggest generally low and/or restricted patterns of expression, and also reveal examples of complex alternative processing, especially in brain, that may have important implications for regulation of protein function. Analysis of complete gene structures of the known genes identified a number of very large introns, a number of very short intergenic distances, and at least one potentially bi-directional promoter. At least 3/4 of known genes and 1/2 of predicted genes are associated with CpG islands. For novel genes, three cases of overlapping genes are predicted. Results of these analyses illustrate some of the complexities inherent in mammalian genome organization and some of the limitations of current sequence analysis technologies. They also doubled the number of potential genes within the region.

Adult↗
PubMed1989

Krüppel requirement for knirps enhancement reflects overlapping gap gene activities in the Drosophila embryo.

Segmental pattern formation in Drosophila proceeds in a hierarchical manner whereby the embryo is stepwise divided into progressively finer regions until it reaches its final metameric form. Maternal genes initiate this process by imparting on the egg a distinct antero-posterior polarity and by directing from the two polar centres the activities of the zygotic genes. The anterior system is strictly dependent on the product of the maternal gene bicoid (bcd), without which all pattern elements in the anterior region of the embryo fail to develop. The posterior system seems to lack such a morphogen. Rather, the known posterior maternal determinants simply define the boundaries within which abdominal segmentation can occur, and the process that actively generates the abdominal body pattern may be entirely due to the interactions between the zygotic genes. The most likely candidates among the zygotic genes that could fulfil the role of initiating the posterior pattern-forming process are the gap genes, as they are the first segmentation genes to be expressed in the embryo. Here we describe the interactions between the gap genes Krüppel (Kr), knirps (kni) and tailless (tll). We show that kni expression is repressed by tll activity, whereas it is directly enhanced by Kr activity. Thus, Kr activity is present throughout the domain of kni expression and forms a long-range protein gradient, which in combination with kni activity is required for abdominal segmentation of the embryo.

Animals↗
PubMed1995

Transfer RNA editing in land snail mitochondria.

Some mitochondrial tRNA genes of land snails show mismatches in the acceptor stems predicted from their gene sequences. The majority of these mismatches fall in regions where the tRNA genes overlap with adjacent downstream genes. We have synthesized cDNA from four circularized tRNAs and determined the sequences of the 5' and 3' parts of their acceptor stems. Three of the four tRNAs differ from their corresponding genes at a total of 13 positions, which all fall in the 3' part of the acceptor stems as well as the discriminator bases. The editing events detected involve changes from cytidine, thymidine, and guanosine to adenosine residues, which generally restore base-pairing in the stems. However, in one case an A-A mismatch is created from an A-C mismatch. It is suggested that this form of RNA editing may involve polyadenylylation of the maturing tRNAs as an intermediate.

Animals↗
PubMed2003

Gene fusion and overlapping reading frames in the mammalian genes for 4E-BP3 and MASK.

4E-BP3 is a member of the eukaryotic initiation factor (eIF) 4F-binding protein family of translational repressors. eIF4E-binding proteins (4E-BPs) inhibit translation initiation by sequestering eIF4E, the cap-binding protein, from eIF4G thus preventing ribosome recruitment to the mRNA. Previous analysis of 4E-BP3 expression uncovered an 8.5-kb mRNA variant of unknown origin. To study this splice variant, we determined the structure of the genomic locus encoding human 4E-BP3 (EIF4EBP3). EIF4EBP3 is located on human chromosome 5q31.3 and comprises three exons (A, B, and C) and two introns. Exon B contains the region of the open reading frame responsible for eIF4E binding. GenBank searches revealed multiple expressed sequence tags originating from the alternative splicing of exon B with unidentified upstream exons. Further studies revealed that the 8.5-kb transcript arises from the fusion of EIF4EBP3 with the mammalian homologue of Drosophila MASK (multiple ankyrin repeats, single KH domain), which is crucial for photoreceptor differentiation, cell survival, and proliferation. Surprisingly, the open reading frame of the MASK-BP3 transcript is different from that of 4E-BP3, which indicates that exon B is translated using an alternative reading frame. A gene fusion similar to that of MASK and EIF4EBP3 has been reported only once in mammals for the UEV1-Kua transcript. The use of an alternative reading frame is also very rare, having been described for two loci, INK4a/ARF and XLalphas/ALEX. The simultaneous exploitation of both mechanisms underscores the flexibility of mammalian genomes and has important implications for the functional analysis of 4E-BP3 and MASK. Interestingly, both eIF4E and MASK are downstream effectors of the Ras/MAPK pathway, which provides a rationale for the MASK-BP3 fusion in mammals.

Alternative Splicing↗
PubMed2003

Identification of a novel gene and a common variant associated with uric acid nephrolithiasis in a Sardinian genetic isolate.

Uric acid nephrolithiasis (UAN) is a common disease with an established genetic component that presents a complex mode of inheritance. While studying an ancient founder population in Talana, a village in Sardinia, we recently identified a susceptibility locus of approximately 2.5 cM for UAN on 10q21-q22 in a relatively small sample that was carefully selected through genealogical information. To refine the critical region and to identify the susceptibility gene, we extended our analysis to severely affected subjects from the same village. We confirm the involvement of this region in UAN through identical-by-descent sharing and autozygosity mapping, and we refine the critical region to an interval of approximately 67 kb associated with UAN by linkage-disequilibrium mapping. After inspecting the genomic sequences available in public databases, we determined that a novel gene overlaps this interval. This gene is divided into 15 exons, spanning a region of approximately 300 kb and generating at least four different proteins (407, 333, 462, and 216 amino acids). Interestingly, the last isoform was completely included in the 67-kb associated interval. Computer-assisted analysis of this isoform revealed at least one membrane-spanning domain and several N- and O-glycosylation consensus sites at N-termini, suggesting that it could be an integral membrane protein. Mutational analysis shows that a coding nucleotide variant (Ala62Thr), causing a missense in exon 12, is in strong association with UAN (P=.0051). Moreover, Ala62Thr modifies predicted protein secondary structure, suggesting that it may have a role in UAN etiology. The present study underscores the value of our small, genealogically well-characterized, isolated population as a model for the identification of susceptibility genes underlying complex diseases. Indeed, using a relatively small sample of affected and unaffected subjects, we identified a candidate gene for multifactorial UAN.

Amino Acid Sequence↗
PubMed1995

Nucleotide sequence and gene organization of the starfish Asterina pectinifera mitochondrial genome.

The 16,260-bp mitochondrial DNA (mtDNA) from the starfish Asterina pectinifera has been sequenced. The genes for 13 proteins, two rRNAs and 22 tRNAs are organized in an extremely economical fashion, similar to those of other animal mtDNAs, with some of the genes overlapping each other. The gene organization is the same as that for another echinoderm, sea urchin, except for the inversion of a 4.6-kb segment that contains genes for two proteins, 13 tRNAs and the 16S rRNA. Judging from the organization of the protein coding genes, mammalian mtDNAs resemble the sea urchin mtDNA more than that of the starfish. The region around the 3' end of the 12S rRNA gene of the starfish shows a high similarity with those for vertebrates. This region encodes a possible stem and loop structure; similar potential structures occur in this region of vertebrate mtDNAs and also in nonmitochondrial small subunit rRNA. A similar stem and loop structure is also found at the 3' end of the 16S rRNA genes in A. pectinifera, in another starfish Pisaster ochraceus, in vertebrates and in Drosophila, but not in sea urchins. The full sequence data confirm the presumption that AGA/AGG, AUA and AAA codons, respectively, code for serine, isoleucine, and asparagine in the starfish mitochondria, and that AGA/AGG codons are read by tRNA(GCUSer), which possesses a truncated dihydrouridine arm, that was previously suggested from a partial mtDNA sequence. The structural characteristics of tRNAs and possible mechanisms for the change in the mitochondrial genetic code are also discussed.

Amino Acid Sequence↗
PubMed2000

Complete sequence of the mitochondrial DNA of the primitive opisthobranch gastropod Pupa strigosa: systematic implication of the genome organization.

The complete sequence (14,189 bp) of the mitochondrial DNA of the opisthobranch gastropod Pupa strigosa was determined. The genome contains 13 protein, 2 rRNA, and 22 tRNA genes typical of metazoan mtDNA. The Pupa mitochondrial genome is highly compact and shows the following unusual features, like pulmonate land snails: (1) extremely small genome size, (2) absence of lengthy noncoding regions (with the largest intergenic spacer being only 46 nt), (3) size reduction of encoded genes, and (4) many overlapping genes. Several tRNA genes exhibit bizarre secondary structures with reduced T or D stems, and many tRNA genes have unstable acceptor stems that might be corrected by posttranscriptional RNA editing. The Pupa mitochondrial gene arrangement is almost identical to those of pulmonate land snails but is radically divergent from those of the prosobranch gastropod Littorina saxatilis and other molluscs. Our finding that the unique gene arrangement and highly compact genome organization are shared between opisthobranch and pulmonate gastropods strongly suggests their close phylogenetic affinity.

Amino Acid Sequence↗
PubMed1990

Analysis of the nucleotide sequence of DNA from the region of the thymidine kinase gene of infectious laryngotracheitis virus; potential evolutionary relationships between the herpesvirus subfamilies.

We have sequenced a 5.4 kb region of the genome from the avian herpesvirus infectious laryngotracheitis virus (ILTV) and identified genes homologous to the thymidine kinase (TK) gene, the capsid p40 gene of herpes simplex virus type 1 (HSV-1), a gene encoding a protein antigenic in Epstein-Barr virus infections plus one other highly conserved gene encoding a protein implicated in cell fusion in HSV-1. Computer analysis of the TK gene and the upstream overlapping gene sequence is used to produce trees showing potential evolutionary relationships between the herpesvirus subfamilies.

Amino Acid Sequence↗
PubMed1997

Molecular cloning and sequence analysis of the lysR gene from the extremely thermophilic eubacterium, Thermus thermophilus HB27.

We have isolated a lysine-auxotrophic and kanamycin-resistant mutant from an extreme thermophile, Thermus thermophilus HB27. This mutant showed the lysA- or lysR- genotype since it could not grow on the minimal plate which contained diaminopimelic acid. Sequence analysis of the clones which could rescue the Lys- mutant indicated the lysR gene. The lysR gene overlapped with the rimK gene for the modification enzyme of ribosomal protein S6. In the Lys- mutant, the lysR gene was disrupted and the C-terminus region of the RimK protein was different from that of the wild-type, which contributed to the Lys- and kanamycin-resistant phenotype. The deduced amino acid sequence of the lysR gene showed 20.9% identity with the LysR protein of Escherichia coli. The percentage of use of cytosine or guanine in the third letter of the codons in the lysR gene was only 67.4%. We also determined that the argC gene encoding N-acetyl-gamma-glutamyl phosphate reductase and the argB gene encoding acetylglutamate kinase were located immediately upstream of the lysR gene.

Anti-Bacterial Agents↗