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PubMed1994

Transcriptional analysis of the region of the herpes simplex virus type 1 genome containing the UL8, UL9, and UL10 genes and identification of a novel delayed-early gene product, OBPC.

The region of the UL component of the herpes simplex virus type 1 genome between nucleotides 17,793 and 25,150 includes three open reading frames that code for the protein products of the UL8, UL9, and UL10 genes (D.J. McGeogh, M.A. Dalrymple, A.J. Davison, A. Dolan, M.C. Frame, D. McNab, L.J. Perry, J.E. Scott, and P. Taylor, J. Gen. Virol. 69:1531-1574, 1988). We have mapped and characterized the overlapping transcripts in this region and have found that, in addition to the low-abundance UL8 and UL9 transcripts and the abundant UL10 transcript, at least two additional transcription units, designated UL8.5 and UL9.5, are specified by this region of the genome. The 5' ends of the UL8, UL8.5, and UL9 transcripts were mapped to nucleotides 20,682, 22,351, and 23,381, respectively. The 5' terminus of the UL9.5 transcript has not yet been mapped. The 3' ends of the UL8, UL8.5, UL9, and UL9.5 transcripts are coterminal at nucleotide 18,197. The 5' end of the UL10 mRNA, which is transcribed from the strand opposite that specifying the UL8, UL8.5, UL9, and UL9.5 transcripts, lies within the UL9 open reading frame at nucleotide 22,944, while the 3' terminus was mapped to nucleotide 24,666. Time course studies demonstrated that the UL8 and UL9 transcripts are members of the early kinetic class, the UL8.5 mRNA is a delayed-early transcript, and the UL9.5 and UL10 transcripts belong to the true-late kinetic class. Examination of the nucleotide sequence of the UL8.5 transcript revealed a potential open reading frame that overlaps and is in frame with the C-terminal half of the open reading frame encoding the origin-binding protein (OBP), the product of the UL9 gene. In vitro translation of the UL8.5 transcript demonstrated that it encodes a protein with an apparent molecular mass of 53 kDa. This protein was recognized by antibody directed against the C-terminal region of OBP and has thus been designated OBPC. A protein with an identical apparent molecular mass was also recognized by this antibody in infected-cell lysates, indicating that OBPC is synthesized during viral infection.

Animals↗
PubMed2004

A genetic variation map for chicken with 2.8 million single-nucleotide polymorphisms.

We describe a genetic variation map for the chicken genome containing 2.8 million single-nucleotide polymorphisms (SNPs). This map is based on a comparison of the sequences of three domestic chicken breeds (a broiler, a layer and a Chinese silkie) with that of their wild ancestor, red jungle fowl. Subsequent experiments indicate that at least 90% of the variant sites are true SNPs, and at least 70% are common SNPs that segregate in many domestic breeds. Mean nucleotide diversity is about five SNPs per kilobase for almost every possible comparison between red jungle fowl and domestic lines, between two different domestic lines, and within domestic lines--in contrast to the notion that domestic animals are highly inbred relative to their wild ancestors. In fact, most of the SNPs originated before domestication, and there is little evidence of selective sweeps for adaptive alleles on length scales greater than 100 kilobases.

Alleles↗
PubMed1993

Nucleotide sequence analysis and epitope mapping of the merozoite surface protein 1 from Plasmodium chabaudi chabaudi AS.

The complete nucleotide sequence of the gene encoding the merozoite surface protein 1 (MSP-1) from the rodent malaria parasite Plasmodium chabaudi chabaudi AS has been determined by direct sequencing of overlapping PCR derived fragments. Comparison of the P. c. chabaudi AS nucleotide sequence with the previously published P. c. chabaudi IP-PC1 sequence indicates that although the MSP-1 gene of these two P. c. chabaudi strains is highly conserved, with sequence identity often approaching 100%, interspersed throughout the molecule are 5 regions of divergence. This is at variance with published data which suggested that the P. c. chabaudi AS and P. c. chabaudi IP-PC1 MSP-1 sequences are largely identical. Epitope mapping studies with a panel of anti-P. c. chabaudi AS MSP-1 monoclonal antibodies demonstrate that whilst most of these mAbs recognise epitopes at the N-terminus of the MSP-1 molecule, two mAbs, including one capable of inhibiting challenge infections in mice in an in vivo passive transfer assay, recognise epitopes which map to the C-terminus.

Amino Acid Sequence↗
PubMed1999

Expression of the open reading frame 74 (G-protein-coupled receptor) gene of Kaposi's sarcoma (KS)-associated herpesvirus: implications for KS pathogenesis.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) encodes a G-protein-coupled receptor (GCR) homolog. This protein is a potent, constitutively active signalling molecule that can influence both proliferation and angiogenesis when ectopically expressed in fibroblasts in vitro. Here we have examined the expression of the KSHV GCR gene in virus-infected lymphoid cells and in KS tumors. Our results show that in both situations the gene is expressed primarily during lytic replication; its transcription is unaffected by inhibition of viral DNA synthesis, indicating that it is expressed in the early phases of the lytic program. The major transcript bearing GCR sequences is bicistronic, harboring coding sequences for another viral gene, K14, at its 5' end. Extensive searches for monocistronic GCR mRNAs using nuclease mapping and reverse transcription-PCR failed to detect such species. The 5' end of K14/GCR mRNA maps to nucleotide (nt) 127848, and its poly(A) addition site maps to nt 130546; a 149-nt intron is present in the K14/GCR intergenic region. These results suggest that the KSHV GCR is translated by unconventional mechanisms involving either translational reinitiation, internal ribosomal entry, or leaky ribosomal scanning. The restriction of GCR expression to the lytic cycle has important implications for the potential role(s) of the GCR in KS pathogenesis.

5' Untranslated Regions↗
PubMed1996

Antagonists of cyclic nucleotide-gated channels and molecular mapping of their site of action.

Activation of photoreceptor and olfactory cyclic nucleotide-gated (CNG) channels involves distinct ligand-binding and channel-gating reactions. To dissociate binding from gating, we identified the first competitive antagonists of CNG channels: specific phosphorothioate derivatives of cAMP and cGMP. We also identified membrane-permeant forms of these molecules that are antagonists and that will be useful for elucidating physiological roles for CNG channels in intact cells. The photoreceptor and olfactory CNG channels determine which of the phosphorothioate derivatives are agonists and which are antagonists based on different structural features of the ligand. The photoreceptor channel uses the nature of the purine ring (adenine vs guanine), whereas the olfactory channel uses the isomeric position of the thiophosphate S atom (Rp vs Sp). Interestingly, the same ligand, Rp-cGMPS, has opposite effects on the two channels, activating the photoreceptor channel and antagonizing the olfactory channel. Because Rp-cGMPS binds to both channels but activates only one, the channels must differ in a protein region that couples binding to gating. Chimeric photoreceptor and olfactory CNG channels reveal that the cytoplasmic C-terminal domain determines whether bound ligand activates the channel successfully. Hence, the C terminus contains not only the cyclic nucleotide-binding site, but also a region that couples ligand binding to channel gating.

Animals↗
PubMed1982

Molecular biology of cell division.

Different domains of the SV40 A gene have different functions, such as viral DNA replication, cell DNA replication, and stimulation of cellular RNA synthesis. The sequences in the SV40 A gene that are critical for the induction of cell DNA synthesis lie on the map between nucleotide 4360 and nucleotide 4001, a stretch of 360 nucleotides coding for 120 of the 708 amino acids of the large T antigen. The sequences critical for stimulation of rRNA synthesis lie on the map further downstream, between nucleotides 3827 and 3506, thus indicating that the signals for growth in size and for cell DNA replication can be dissociated. Methylation of the SV40 A gene at multiple ECoRI* sites has no effect on its expression. However, methylation of the HSV-TK gene at one single ECoRI site 70 base pairs upstream from the cap site inhibits its expression. The results indicate that methylation of genes affects their expression, but only when methylation occurs at specific sites.

Amanitins↗
PubMed2001

A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms.

We describe a map of 1.42 million single nucleotide polymorphisms (SNPs) distributed throughout the human genome, providing an average density on available sequence of one SNP every 1.9 kilobases. These SNPs were primarily discovered by two projects: The SNP Consortium and the analysis of clone overlaps by the International Human Genome Sequencing Consortium. The map integrates all publicly available SNPs with described genes and other genomic features. We estimate that 60,000 SNPs fall within exon (coding and untranslated regions), and 85% of exons are within 5 kb of the nearest SNP. Nucleotide diversity varies greatly across the genome, in a manner broadly consistent with a standard population genetic model of human history. This high-density SNP map provides a public resource for defining haplotype variation across the genome, and should help to identify biomedically important genes for diagnosis and therapy.

Chromosome Mapping↗
PubMed1991

The primary structure of the thymidine kinase gene of fish lymphocystis disease virus.

The DNA nucleotide sequence of the thymidine kinase (TK) gene of fish lymphocystis disease virus (FLDV) which has been localized between the coordinates 0.678 to 0.688 of the viral genome was determined. The analysis of the DNA nucleotide sequence located between the recognition sites of HindIII (0.669 map unit; nucleotide position 1) and AccI (nucleotide position 2032) revealed the presence of an open reading frame of 954 bp on the lower strand of this region between nucleotide positions 1868 (ATG) and 915 (TAA). It encodes for a protein of 318 amino acid residues. The evolutionary relationships of the TK gene of FLDV to the other known TK genes was investigated using the method of progressive sequence alignment. These analyses revealed a high degree of diversity between the protein sequence of FLDV TK gene and the amino acid composition of other TKs tested. However, significant conservations were detected at several regions of amino acid residues of the FLDV TK protein when compared to the amino acid sequence of TKs of African swine fever virus, fowlpox virus, shope fibroma virus, and vaccinia virus and to the amino acid sequences of the cellular cytoplasmic TK of chicken, mouse, and man.

Amino Acid Sequence↗
PubMed1986

Complete nucleotide sequence and mRNA-mapping of the large subunit gene of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Chlamydomonas moewusii.

Nucleotide (nt) sequence of the large subunit (LS) gene of ribulose-1,5-bisphosphate carboxylase/oxygenase from the green alga, Chlamydomonas moewusii, and mapping of transcription ends was achieved by two new strategies. The deduced LS sequence of 475 amino acid residues was compared with similar genes from six other species; cyanobacteria, land plants and a related alga (C. reinhardtii). The most conserved regions are the three ribulose bisphosphate binding sites and the CO2 activator site. The nt sequence conservation outside the coding region is limited to only three segments within the 5'-flanking region: a region of tandem repeats, TATAA box and ribosome-binding site. Termination point of transcription is an 'A' residue 3' to the first of two 18-nt inverted repeats, which has the potential to form a stem-loop hairpin structure. The possible role of these potential regulatory features for transcription and translation, and similar structures in other LS genes is presented.

Amino Acid Sequence↗
PubMed2006

The human growth hormone gene contains a silencer embedded within an Alu repeat in the 3'-flanking region.

Alu family sequences are middle repetitive short interspersed elements (SINEs) dispersed throughout vertebrate genomes that can modulate gene transcription. The human (h) GH locus contains 44 complete and four partial Alu elements. An Sx Alu repeat lies in close proximity to the hGH-1 and hGH-2 genes in the 3'-flanking region. Deletion of the Sx Alu repeat in reporter constructs containing hGH-1 3'-flanking sequences increased reporter activity in transfected pituitary GC cells, suggesting this region contained a repressor element. Analysis of multiple deletion fragments from the 3'-flanking region of the hGH-1 gene revealed a strong orientation- and position-independent silencing activity mapping between nucleotides 2158 and 2572 encompassing the Sx Alu repeat. Refined mapping revealed that the silencer was a complex element comprising four discrete entities, including a core repressor domain (CRD), an antisilencer domain (ASE) that contains elements mediating the orientation-independent silencer activity, and two domains flanking the CRD/ASE that modulate silencer activity in a CRD-dependent manner. The upstream modulator domain is also required for orientation-independent silencer function. EMSA with DNA fragments representing all of the silencer domains yielded a complex pattern of DNA-protein interactions indicating that numerous GC cell nuclear proteins bind specifically to the CRD, ASE, and modulator domains. The silencer is GH promoter dependent and, in turn, its presence decreases the rate of promoter-associated histone acetylation resulting in a significant decrease of RNA polymerase II recruitment to the promoter. The silencer may provide for complex regulatory control of hGH gene expression in pituitary cells.

Alu Elements↗
PubMed1990

Characterization and distribution of an interspersed repeated nucleotide sequence from Lophopyrum elongatum and mapping of a segregation-distortion factor with it.

Repeated nucleotide sequence pLeUCD2 cloned from Lophopyrum elongatum is highly abundant in the genomes of diploid and polyploid wheatgrass species of genera Lophopyrum, Thinopyrum, Pseudoroegneria, Agropyron, Elymus, Elytrigia, and Pascopyrum but undetectable by Southern blot hybridization in Triticum and representative species of Dasypyrum, Hordeum, Psathyrostachys, Secale, Taeniatherum, Heteranthelium, and Leymus in the tribe Triticeae. The DNA fragment inserted in pLeUCD2 is 277 bp long and AT rich (65%), and contains numerous inverted and palindromic repeats. In situ DNA hybridization substantiated a previous hypothesis that the sequence is interspersed in the wheatgrass genomes. Heterogeneity and clustering of like repeats of the pLeUCD2 family along wheatgrass chromosomes was used to map a segregation-distortion factor, designated Sd-1, proximal to the Lr19 locus in recombinant chromosomes of L. ponticum chromosome 7Ag and wheat chromosome 7D.

Base Sequence↗
PubMed1985

Nucleotide sequence of a major class-III phage-T3 RNA-polymerase promoter located at 98.0% of phage-T3 genetic map.

The entire nucleotide sequence of a 409-bp HincII fragment, located within the MboI-E fragment on bacteriophage T3 DNA and containing a major class-III T3 RNA polymerase promoter positioned at 98% on the standard T3 genetic map, has been determined. Alignment of this class-III promoter with previously determined T3 RNA polymerase promoters, with start points of transcription (+1) in register, indicates high degree of sequence conservation between position -16 to +6 among all T3 RNA polymerase promoters. The conserved portion of the (-) strand sequence is 5'-A-TA-T-AT-A-C-C-C-T-C-A-C-T-A-A-A-G-G-G-A---3'. This fragment also contains an open reading frame (ORF) with a translational start codon located at position +146 which is preceded by a potential ribosome binding site (RBS). There is more than 70% amino acid-sequence homology between the deduced sequences of the -NH2 terminal region of this putative T3 phage protein and the corresponding protein coded by bacteriophage T7 (protein of T7 gene 19.5).

Base Sequence↗
PubMed1991

In vivo mapping of a DNA adduct at nucleotide resolution: detection of pyrimidine (6-4) pyrimidone photoproducts by ligation-mediated polymerase chain reaction.

DNA adducts in unique sequences along the mammalian genome are mapped in vivo at single-nucleotide resolution. Pyrimidine (6-4) pyrimidone photoproducts [(6-4) photoproducts] represent one of the two major adduct classes found after UV irradiation of DNA and were shown to play an important role in UV-induced mutagenesis. After UV light treatment of cells, DNA is prepared and chemically cleaved at (6-4) photoproducts with piperidine. Gene-specific fragments are then amplified from total genomic DNA by use of a ligation-mediated polymerase chain reaction. Analysis of the human chromosome X-linked phosphoglycerate kinase (PGK1) gene's promoter has shown that the frequency of (6-4) photoproducts expressed as piperidine-labile sites is (i) high at TpC and CpC dinucleotides, (ii) dependent on the nearest-neighbor bases, (iii) inhibited by the binding of a transcription factor, and (iv) different for DNA derived from the active and inactive X chromosome. This latter difference is mainly a consequence of the presence of 5-methylcytosine (m5C) in CpG dinucleotides on the inactive X chromosome. 5-Methylcytosine in the sequences Tm5CG and Cm5CG inhibits the formation of (6-4) photoproducts. Thus, in addition to in vivo mapping of a DNA adduct at nucleotide resolution, we also report another method for methylation analysis and photofootprinting.

Animals↗
PubMed1989

Intraspecific evolution of a gene family coding for urinary proteins.

The genome of the laboratory mouse contains about 35 major urinary protein (MUP) genes, many of which are clustered on chromosome 4. We have used distance and parsimony methods to estimate phylogenetic relationships between MUP genes from nucleotide sequence and restriction maps. By analyzing coding sequences we show that the genes fall into four main groups of related sequences (groups 1-4). Comparisons of restriction maps and the nucleotide sequences of hypervariable regions that lie 50 nucleotides 5' to the cap sites show that the group 1 genes and probably also the group 2 pseudogenes fall into subgroups. The most parsimonious trees are consistent with the evolution of the array of group 1 and 2 genes by mutation accompanied by a process tending toward homogenization such as unequal crossing-over or gene conversion. The phylogenetic grouping correlates with grouping according to aspects of function. The genomes of the inbred strains BALB/c and C57BL contain different MUP gene arrays that we take to be samples from the wild population of arrays.

Animals↗
PubMed1988

Genomic mapping by fingerprinting random clones: a mathematical analysis.

Results from physical mapping projects have recently been reported for the genomes of Escherichia coli, Saccharomyces cerevisiae, and Caenorhabditis elegans, and similar projects are currently being planned for other organisms. In such projects, the physical map is assembled by first "fingerprinting" a large number of clones chosen at random from a recombinant library and then inferring overlaps between clones with sufficiently similar fingerprints. Although the basic approach is the same, there are many possible choices for the fingerprint used to characterize the clones and the rules for declaring overlap. In this paper, we derive simple formulas showing how the progress of a physical mapping project is affected by the nature of the fingerprinting scheme. Using these formulas, we discuss the analytic considerations involved in selecting an appropriate fingerprinting scheme for a particular project.

Animals↗
PubMed1991

Advances in DNA sequencing technology.

Efforts to map and sequence the genomes of the human and other species have stimulated efforts to improve the technology required for these endeavors. During the last year, these efforts have produced substantial advances in DNA template preparation, sequencing chemistry, and gel analysis.

Base Sequence↗
PubMed1990

On the possibility of obtaining a physical map of genomes by photoelectron imaging.

Photoelectron imaging provides the possibility of a new method of mapping chromosomes. The basic concept is to cause DNA to emit electrons under the action of UV light. The criteria which must be met to map genomes by photoelectron imaging are set forth and discussed. Forming an image of the DNA by accelerating and focusing the electrons is a necessary but not sufficient condition for genome mapping. Equally important is to identify wavelengths of UV light which will cause selective emission from the base pairs, adenine-thymine and guanine-cytosine. The resulting image would then contain a modulation in the image brightness along the DNA duplex. By examining the photoelectron current from uniform films of homopolymers, a wavelength region is identified where marked differences in emission from base pairs is observed. At 160 nm, for example, the relative electron emission from a film of poly(dGdC) is approximately 5 times greater than for an equivalent film of poly(dAdT). Using the experimental data and known sequences, photoelectron gene maps are calculated for the bacteriophage lambda and for a short interspersed repetitive DNA sequence (an Alu repeat) of the human genome. The results suggest that a 5-nm physical map of chromosomes generated by photoelectron imaging would be informative and useful in mapping human and other large genomes.

Bacteriophage lambda↗