Identification of a conserved sequence in the non-coding regions of many human genes.

We have analyzed a sequence of approximately 70 base pairs (bp) that shows a high degree of similarity to sequences present in the non-coding regions of a number of human and other mammalian genes. The sequence was discovered in a fragment of human genomic DNA adjacent to an integrated hepatitis B virus genome in cells derived from human hepatocellular carcinoma tissue. When one of the viral flanking sequences was compared to nucleotide sequences in GenBank, more than thirty human genes were identified that contained a similar sequence in their non-coding regions. The sequence element was usually found once or twice in a gene, either in an intron or in the 5' or 3' flanking regions. It did not share any similarities with known short interspersed nucleotide elements (SINEs) or presently known gene regulatory elements. This element was highly conserved at the same position within the corresponding human and mouse genes for myoglobin and N-myc, indicating evolutionary conservation and possible functional importance. Preliminary DNase I footprinting data suggested that the element or its adjacent sequences may bind nuclear factors to generate specific DNase I hypersensitive sites. The size, structure, and evolutionary conservation of this sequence indicates that it is distinct from other types of short interspersed repetitive elements. It is possible that the element may have a cis-acting functional role in the genome.     

Sequence analysis of members of the human Ig VH4 gene family derived from a single VH locus. Identification of novel germ-line members.

We have generated a mouse x human heterohybridoma that contains a single copy of chromosome 14 and, thus, a haploid set of Ig VH genes. This cell line was used to investigate the germ-line content and nucleotide sequences of members of the VH4 gene family in a polymerase chain reaction-based approach. The analysis of 58 full-length sequences revealed the presence of 12 different germ-line VH4 genes, each of which is potentially functional. These germ-line VH4 genes were compared with the nucleotide sequences of published VH4 genes. Three VH4 genes were 100% identical to previously published sequences and belong to a group of VH4 genes that are strongly conserved and highly prevalent in the human population. Three VH4 genes in our collection displayed greater than 99.3% sequence identity with reported germ-line VH4 sequences and likely represent allelic counterparts of these genes. Six genes displayed less than 97.2% sequence identity with published VH4 genes and were identified as novel members of the human VH4 gene family or more distantly related alleles of known VH4 genes. Collectively, these data suggest that, overall, the human VH4 gene family may be more diverse than hitherto assumed, whereas a number of individual members are nonpolymorphic and extremely well conserved.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

Genes of the lipase family: comparison of nucleic and proteinic sequences]

Vertebrates' plasmatic apolipoproteins and a few number of lipases in their metabolism present sequence homologies. They are grouped in genes families. The four exons apolipoproteins gene family includes nine human genes: the divergence rate of their sequences allows to place the first ancestral gene very high in the phylogenetic tree of the evolution. However, a more recent duplication of apolipoprotein C-I gene dating from 40 millions years, may be a phylogenetic marker for the radiation of Monkeys. Pancreatic lipase and isoforms, lipoprotein-lipase and hepatic triacylglycerol-lipase form by their homologies a "superfamily" of genes, which also includes yolk proteins of Dipterians eggs. Sequence homologies of PL, LPL and HL are analysed and compared with multiple alignments of amino-acids and nucleotides on spreadsheets. From these comparisons we may characterize four classes of phylogenetic markers: 1) repetitive DNA sequence (Alu, B1, PRE-1) appeared during Mammals evolution, 2) short insertions or deletions (within N-terminal domain) and a gene conversion in guinea-pig lineage, 3) a progressive reduction of intron number during the lipases evolution, 4) several duplications of genes which have produced the five genes of this superfamily currently known in the human genome.     

Comparative gene finding in chicken indicates that we are closing in on the set of multi-exonic widely expressed human genes

The recent availability of the chicken genome sequence poses the question of whether there are human protein-coding genes conserved in chicken that are currently not included in the human gene catalog. Here, we show, using comparative gene finding followed by experimental verification of exon pairs by RT–PCR, that the addition to the multi-exonic subset of this catalog could be as little as 0.2%, suggesting that we may be closing in on the human gene set. Our protocol, however, has two shortcomings: (i) the bioinformatic screening of the predicted genes, applied to filter out false positives, cannot handle intronless genes; and (ii) the experimental verification could fail to identify expression at a specific developmental time. This highlights the importance of developing methods that could provide a reliable estimate of the number of these two types of genes.     

Genome Duplications and Other Features in 12 Mb of DNA Sequence from Human Chromosome 16p and 16q

Several publicly funded large-scale sequencing efforts have been initiated with the goal of completing the first reference human genome sequence by the year 2005. Here we present the results of analysis of 11.8 Mb of genomic sequence from chromosome 16. The apparent gene density varies throughout the region, but the number of genes predicted (84) suggests that this is a gene-poor region. This result may also suggest that the total number of human genes is likely to be at the lower end of published estimates. One of the most interesting aspects of this region of the genome is the presence of highly homologous, recently duplicated tracts of sequence distributed throughout the p-arm. Such duplications have implications for mapping and gene analysis as well as the predisposition to recurrent chromosomal structural rearrangements associated with genetic disease.     

Mhc-DRB genes of the pigtail macaque (Macaca nemestrina): implications for the evolution of human DRB genes.

The DRB family of human class II major histocompatibility complex (Mhc) loci is unusual in that individuals differ in the number and combination of genes (haplotypes) they carry. Indications are that both the allelic and haplotype polymorphisms of the DRB loci predate speciation. Searching for the evolutionary origins of these polymorphisms, we have sequenced five DRB clones isolated from a cDNA library of a pigtail macaque (Macaca nemestrina) B lymphocyte line. The clones represent five different genes which we designate Mane-DRB*01-Mane-DRB*05. The genes appears to be approximately equidistant from each other, so that allelic relationships between them cannot be established on the basis of the sequence data alone. If positions coding for the peptide-binding region of the class II beta chains are eliminated from sequence comparisons, the Mane-DRB genes appear to be most closely related to the human (HLA) DRB1 genes of the DRw52 group. We interpret this finding to indicate that the ancestral gene of the DRw52 group of human DRB1 alleles separated from the rest of the HLA-DRB1 alleles before the separation of the Old World monkeys (Cercopithecoidea) from the apes (Hominoidea) in the early Oligocene. After this separation, the ancestral DRB1 gene of the DRw52 group duplicated in the Old World monkey lineage to give rise to genes at three loci at least, while in the ape lineage this gene may have remained single and diverged into a number of alleles instead. These findings suggest that some of the polymorphism currently present at the DRB1 locus is greater than 35 Myr old.     

Three expressed sequences within the human beta-tubulin multigene family each define a distinct isotype

This paper describes the isolation and complete sequence of a novel expressed human beta-tubulin gene (beta 2). The sequence is compared with that of two other expressed human beta-tubulin genes (M40 and 5 beta). All are encoded by four exons. Though the boundaries of each exon are absolutely conserved among the three genes, the intervening sequences differ considerably in size and sequence content. Two of the genes (M40 and 5 beta) contain one (M40) or ten (5 beta) members of the middle repetitive Alu family sequences within one of their intervening sequences. Comparison of the amino acid sequences encoded by each gene reveals a high level of homology overall, though there is significant divergence between the carboxy termini of two of the genes. The pattern of expression of each beta-tubulin gene has been studied in several different human cell lines using unique non-crosshybridizing probes derived from the 3' untranslated regions. Two of the genes, M40 and beta 2, are expressed at varying levels in all of the cell lines examined, though the level of expression of one of these genes parallels the other in most cases. The third gene, 5 beta, is detectably expressed only in cells of neural origin. Thus, distinct human beta-tubulin isotypes are encoded by genes whose exon size and number has been conserved evolutionarily, but whose pattern of expression may be regulated either co-ordinately or uniquely. Of the approximately 15 sequences contained in the human beta-tubulin multigene family, nine have now been sequenced fully. The overall composition of the multigene family and the evolutionary relationships among its various members are discussed.     

The importance of alternative splicing in the drug discovery process

The publication of the sequence of the human genome revealed that the gene count in humans is much lower than previously estimated. Although textbooks usually place the number at 100,000, it is currently estimated that the human genome contains no more than 30,000 protein-coding genes. How can the great complexity of human life be explained by this number, which is less than twice the number of genes in the primitive worm C. elegans? The answer probably lies in the recent discovery that about half of all human genes undergo alternative splicing. This paper reviews the broad implications of alternative splicing for the drug-discovery process.     

The human T cell receptor beta-chain gene complex contains at least 57 variable gene segments. Identification of six V beta genes in four new gene families.

The human TCR beta-chain gene complex includes at least 57 variable (V) gene segments, a number estimated using a combination of Southern blots of conventional and pulsed field gels, sequence analysis of cDNA clones, and from the analysis of genomic cosmid and phage clones. This number includes six TCR beta-chain V genes in four new families identified here by sequence analysis of clones derived from a human TCR beta-chain specific cDNA library. Comparison of the sequences of the new V beta genes with previously reported V beta sequences reveals predicted similarities but less than 75% nucleic acid identity that establishes them as new V beta families. One of the new V beta gene families includes three genes and the other three are single member families. Identification of these six new V beta genes falling into four V beta families brings the total number of transcribed human V beta families to 24 and makes it possible to refine the estimate of the total number of human TCR V beta genes to 57.     

Ancient duplications of the human proglucagon gene

The human proglucagon gene (GCG) is encoded within a finished 576-kb DNA sequence generated by the Human Genome Project. GCG is flanked by 18 kb and 65 kb of DNA, 5' and 3', respectively, that do not encode genes. The genomic sequence that includes GCG was found to have a long history of gene duplication events. Some members of the glucagon-like family of genes, GCG on chromosome 2 and GIP on chromosome 17, may be products of ancient genome duplications on the early vertebrate lineage. A large genomic tandem duplication event that included DPP4-like and GCG genes occurred before the amphibian-mammal divergence, but one of the duplicated copies of GCG has been lost on the human lineage. Recently, a processed pseudogene of the X-chromosome-linked gene TIMM8A was inserted downstream of GCG. Some ancient duplicates of GCG may retain physiological functions in other vertebrates.     

Quantitating tissue specificity of human genes to facilitate biomarker discovery

We describe a method to identify candidate cancer biomarkers by analyzing numeric approximations of tissue specificity of human genes. These approximations were calculated by analyzing predicted tissue expression distributions of genes derived from mapping expressed sequence tags (ESTs) to the human genome sequence using a binary indexing algorithm. Tissue-specificity values facilitated high-throughput analysis of the human genes and enabled the identification of genes highly specific to different tissues. Tissue expression distributions for several genes were compared to estimates obtained from other public gene expression datasets and experimentally validated using quantitative RT-PCR on RNA isolated from several human tissues. Our results demonstrate that most human genes ( approximately 98%) are expressed in many tissues (low specificity), and only a small number of genes possess very specific tissue expression profiles. These genes comprise a rich dataset from which novel therapeutic targets and novel diagnostic serum biomarkers may be selected.     

Different structural organization of the encephalopsin gene in man and mouse

Encephalopsin, also called Panopsin, is a recently discovered extraretinal photoreceptor, which may play a role in non-visual photic processes such as the entrainment of circadian rhythm or the regulation of pineal melatonin production. Based on RT-PCR data and comparative genomic sequence analysis, we show that the human OPN3 gene consists of six exons and expresses various splice variants, while the murine homologue contains four exons and produces just one splice form. Furthermore, the human OPN3 gene overlaps with the neighboring KMO gene on a genomic as well as on an RNA level, whereas the corresponding genes in mouse lie close together but do not overlap. This finding is of particular interest, since differences in gene organization between man and mouse, that have been reported so far, occur within gene clusters, i.e. the number of genes within a certain cluster may differ between man and mouse. OPN3 provides an exception to this rule, since it is positionally uncoupled from other genes of the opsin family.     

Complete nucleotide sequence of the influenza A/PR/8/34 virus NS gene and comparison with the NS genes of the A/Udorn/72 and A/FPV/Rostock/34 strains.

The nucleotide sequence of the NS gene of the human influenza virus A/PR/8/34 was determined and found to be the same length (890 nucleotides) as the NS gene of another human influenza virus A/Udorn/72 and of the avian isolate A/FPV/Rostock/34. Comparison of the sequences of the NS genes of the two human influenza viruses shows an 8.9% difference whereas the NS gene of the avian isolate differs by only 8% from that of the human strain A/PR/8/34. The extensive sequence similarity among these three genes does not support the notion of species specific homology groups among NS genes of avian and human influenza virus strains. The primary sequence of the A/PR/8/34 NS gene is consistent with the findings that the influenza virus NS gene may code for two overlapping polypeptides. In addition, an open reading frame potentially coding for a polypeptide 167 amino acids in length was found in the negative strand RNA of the A/PR/8/34 virus NS gene.     

Two mouse early embryonic beta-globin gene sequences. Evolution of the nonadult beta-globins

We have determined the complete nucleotide sequence of two early embryonic beta-globin genes of the BALB/c mouse: beta h0 and beta h1 X beta h1 codes for the embryonic z protein, while the beta h0 gene may be a minor early embryonic beta-globin gene. The general sequence organization of both genes is entirely analogous to other functional globin genes. There is, however, a 220-base pair insertion of unique sequence within the first intron of beta h0 X beta h0 and beta h1 are 96% homologous for 260 base pairs 5' to the AUG initiation codon, and 93% homologous throughout their coding regions. Analysis of the 5'-flanking sequence demonstrates that these genes are more nonadult-like than adult-like. The sequences show evidence for gene conversions among the mouse nonadult beta-globin genes that were limited to individual exons, presumably by the presence of non-homologous introns. We propose that this arrangement has the beneficial evolutionary effect of allowing gene conversion to act independently on regions of the protein with different structural or functional responsibilities. beta h0 and beta h1 are evolutionary homologs to the human fetal and rabbit beta 3 genes, while their manner of expression is similar to rabbit beta 3 and dissimilar to human fetal expression. The evolutionary history of the human beta-globin genes, therefore, includes the recruitment of an embryonic gene to fetal developmental control.