Sequence, structure, and evolution of a complete human olfactory receptor gene cluster

The olfactory receptor (OR) gene cluster on human chromosome 17p13.3 was subjected to mixed shotgun automated DNA sequencing. The resulting 412 kb of genomic sequence include 17 OR coding regions, 6 of which are pseudogenes. Six of the coding regions were discovered only upon genomic sequencing, while the others were previously reported as partial sequences. A comparison of DNA sequences in the vicinity of the OR coding regions revealed a common gene structure with an intronless coding region and at least one upstream noncoding exon. Potential gene control regions including specific pyrimidine:purine tracts and Olf-1 sites have been identified. One of the pseudogenes apparently has evolved into a CpG island. Four extensive CpG islands can be discerned within the cluster, not coupled to specific OR genes. The cluster is flanked at its telomeric end by an unidentified open reading frame (C17orf2) with no significant similarity to any known protein. A high proportion of the cluster sequence (about 60%) belongs to various families of interspersed repetitive elements, with a clear predominance of LINE repeats. The OR genes in the cluster belong to two families and seven subfamilies, which show a relatively high degree of intermixing along the cluster, in seemingly random orientations. This genomic organization may be best accounted for by a complex series of evolutionary events.     

Structure of the murine lactotransferrin gene is similar to the structure of other transferrin-encoding genes and shares a putative regulatory region with the murine myeloperoxidase gene

The structure and nucleotide sequence of the murine lactotransferrin-encoding gene (LTF) deduced partly by direct sequencing of genomic clones in the λ phage vector and partly by enzymatic amplification of genomic DNA segments primed with the oligodeoxyribonucleic primers homologous to the cDNA sequence. The λ phage clones contained the 5′ half of the gene corresponding to the first eight exons and an incomplete ninth exon interrupted by eight introns. Genomic clones corresponding to the 3′ half of the LTF gene could not be obtained on repeated attempts from two different mouse genomic libraries, suggesting the possible presence of unclonable sequences in this part of the gene. Hence, PCR was used to clone the rest of the gene. Four out of the presumed eight remaining introns were cloned along with the flanking exons using PCR. Comparison of the structure of the LTF gene with those of the two other known transferrin-encoding genes, human serum transferrin-encoding gene and chicken ovotransferrin-encoding gene reveals that all three genes have a very similar intron-exon distribution pattern. The hypothesis that the present-day transferrin-encoding genes have originated from duplication of a common ancestral gene is confirmed here at the gene level. An interesting finding is the identification of a region of shared nucleotides between the 5′ flanking regions of the murine LTF and myeloperoxidase-encoding genes, the two genes expressed specifically in neutrophilic granulocytes.     

Nucleotide sequence analysis of the delta beta-globin gene region in humans

The continuous DNA sequence of a 16.5-kilobase pair region encompassing the linked delta beta-globin gene cluster in humans is presented with a detailed restriction endonuclease map. There are 38 differences (0.5%) in comparison with published sequence data, corrected for errors in sequencing, resulting in polymorphic rates of 0.2% in exons and 0.76% in 5'-gene flanking regions. Fifteen changes result in the generation or elimination of restriction sites which may be useful in linkage disequilibrium studies. Two pairs of inverted Alu repeats, a pyrimidine-rich region 5' to delta, and (TG)n, (Pu/Py)n, and (ATTTT)n tracts 5' to beta are described. Dinucleotide frequencies and deviation from expected values approximated those found in total human genomic DNA. Regions of less than 50% A + T content were found associated with Alu sequences, a 150-base pair region immediately 5' to the beta gene, exon regions from both genes, and an area 3' to the beta gene. These regions also contained significantly lower than expected CpG levels compared to other regions, suggesting a possible relationship between DNA organizational patterns and functionally important regions. In addition, strand asymmetries in base composition in this region differ from those associated with the fetal globin genes.     

Sequence and structure of the nucleolin promoter in rodents: characterization of a strikingly conserved CpG island

We report the isolation of the complete genes encoding nucleolin from rat and hamster. The DNA clones were obtained from partial genomic libraries by probing with a genomic DNA fragment containing the leader and promoter regions of the mouse nucleolin gene. We have determined the complete nucleotide sequence of the 5'-terminal region for the three rodent species. The sequenced regions extend over 1 kb downstream and upstream from the cap sites and include a conserved CpG island 1500 nucleotides (nt) long. The 5' end of the CpG island in each species has maintained a long alternating purine-pyrimidine sequence which could adopt a Z-DNA conformation. By sequence comparison, 42 blocks of homology are defined in the 5'-terminal region, of which 36 appear in the CpG island and contain numerous conserved CpG dinucleotides. Two blocks, 110 and 49 nt long, encompassing the cap sites and the region immediately upstream, respectively, present features characteristic of regulated genes: a possible TATA box (ATTA), two pyrimidine-rich nucleotide stretches and two inverted juxtaposed CCAAT-like boxes (GGTTGG). Furthermore, the adjacent upstream conserved region presents features characteristic of housekeeping genes: four G/C boxes, embedded in a high G + C-content sequence, among them one presenting a perfect consensus Sp 1-binding site (GCCCCGCCCC). Among unusual features, we report numerous large G + C-rich conserved sequences located in the first intron. One of these sequences contains two G/C boxes which border a sequence presenting a dyad symmetry (GCGCACGTGCTC). Our findings shed some light on the putative role of the CpG island. We show that CpG-rich sequence motifs are under strong selective pressure over the whole 5'-terminal region and are presumably involved in regulatory mechanisms.     

A distinct sequence (ATAAA)n separates methylated and unmethylated domains at the 5'-end of the GSTP1 CpG island

What defines the boundaries between methylated and unmethylated domains in the genome is unclear. In this study we used bisulfite genomic sequencing to map the boundaries of methylation that flank the 5'- and 3'-ends of the CpG island spanning the promoter region of the glutathione S-transferase (GSTP1) gene. We show that GSTP1 is expressed in a wide range of tissues including brain, lung, skeletal muscle, spleen, pancreas, bone marrow, prostate, heart, and blood and that this expression is associated with the CpG island being unmethylated. In these normal tissues a marked boundary was found to separate the methylated and unmethylated regions of the gene at the 5'-flank of the CpG island, and this boundary correlated with an (ATAAA)(19-24) repeated sequence. In contrast, the 3'-end of the CpG island was not marked by a sharp transition in methylation but by a gradual change in methylation density over about 500 base pairs. In normal tissue the sequences on either side of the 5'-boundary appear to lie in separate domains in which CpG methylation is independently controlled. These separate methylation domains are lost in all prostate cancer where GSTP1 expression is silenced and methylation extends throughout the island and spans across both the 5'- and 3'-boundary regions.     

Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the GAP-related domain

More than 500 unrelated patients with neurofibromatosis type 1 (NF1) were screened for mutations in the NF1 gene. For each patient, the whole coding sequence and all splice sites were studied for aberrations, either by the protein truncation test (PTT), temperature-gradient gel electrophoresis (TGGE) of genomic PCR products, or, most often, by direct genomic sequencing (DGS) of all individual exons. A total of 301 sequence variants, including 278 bona fide pathogenic mutations, were identified. As many as 216 or 183 of the genuine mutations, comprising 179 or 161 different ones, can be considered novel when compared to the recent findings of Upadhyaya and Cooper, or to the NNFF mutation database. Mutation-detection efficiencies of the various screening methods were similar: 47.1% for PTT, 53.7% for TGGE, and 54.9% for DGS. Some 224 mutations (80.2%) yielded directly or indirectly premature termination codons. These mutations showed even distribution over the whole gene from exon 1 to exon 47. Of all sequence variants determined in our study, <20% represent C-->T or G-->A transitions within a CpG dinucleotide, and only six different mutations also occur in NF1 pseudogenes, with five being typical C-->T transitions in a CpG. Thus, neither frequent deamination of 5-methylcytosines nor interchromosomal gene conversion may account for the high mutation rate of the NF1 gene. As opposed to the truncating mutations, the 28 (10.1%) missense or single-amino-acid-deletion mutations identified clustered in two distinct regions, the GAP-related domain (GRD) and an upstream gene segment comprising exons 11-17. The latter forms a so-called cysteine/serine-rich domain with three cysteine pairs suggestive of ATP binding, as well as three potential cAMP-dependent protein kinase (PKA) recognition sites obviously phosphorylated by PKA. Coincidence of mutated amino acids and those conserved between human and Drosophila strongly suggest significant functional relevance of this region, with major roles played by exons 12a and 15 and part of exon 16.     

Concurrent evolution of human immunodeficiency virus type 1 in patients infected from the same source: rate of sequence change and low frequency of inactivating mutations.

Direct sequencing of segments of the envelope gene of human immunodeficiency virus type 1 proviruses in peripheral blood mononuclear cells has revealed that a cohort of hemophiliacs who were infected after exposure to a single common batch of factor VIII share closely related virus strains. Seventy-four sequences extending from hypervariable regions V4 through V5 from nine patients yielded a mean intrapatient nucleotide distance of 5.5%, while a mean of 4.2% was observed in 39 sequences of the V3 loop (six patients). Phylogenetic analysis revealed that sequences of six Edinburgh patients were particularly closely related and those from a patient infected in the United States were very distinct. The mean nucleotide distance among these six was 8.3%, while the mean distance from the U.S.-derived sequences was 25.5% in the V4-V5 region. The rate of sequence change across this patient group has been estimated to be 0.4% per year in the V4-V5 region and 0.5% per year in the V3 region, with at least a twofold range across patients. Only two inactivating nucleotide substitutions have been observed in a total of 42 kb of sequence obtained from the env and gag genes during this study.     

Both hypomethylation and hypermethylation in a 0.2-kb region of a DNA repeat in cancer

NBL2 is a tandem 1.4-kb DNA repeat, whose hypomethylation in hepatocellular carcinomas was shown previously to be an independent predictor of disease progression. Here, we examined methylation of all cytosine residues in a 0.2-kb subregion of NBL2 in ovarian carcinomas, Wilms' tumors, and diverse control tissues by hairpin-bisulfite PCR. This new genomic sequencing method detects 5-methylcytosine on covalently linked complementary strands of a DNA fragment. All DNA clones from normal somatic tissues displayed symmetrical methylation at seven CpG positions and no methylation or only hemimethylation at two others. Unexpectedly, 56% of cancer DNA clones had decreased methylation at some normally methylated CpG sites as well as increased methylation at one or both of the normally unmethylated sites. All 146 DNA clones from 10 cancers could be distinguished from all 91 somatic control clones by assessing methylation changes at three of these CpG sites. The special involvement of DNA methyltransferase 3B in NBL2 methylation was indicated by analysis of cells from immunodeficiency, centromeric region instability, and facial anomalies syndrome patients who have mutations in the gene encoding DNA methyltransferase 3B. Blot hybridization of 33 cancer DNAs digested with CpG methylation-sensitive enzymes confirmed that NBL2 arrays are unusually susceptible to cancer-linked hypermethylation and hypomethylation, consistent with our novel genomic sequencing findings. The combined Southern blot and genomic sequencing data indicate that some of the cancer-linked alterations in CpG methylation are occurring with considerable sequence specificity. NBL2 is an attractive candidate for an epigenetic cancer marker and for elucidating the nature of epigenetic changes in cancer.     

Sequence and comparative analysis of the rabbit alpha-like globin gene cluster reveals a rapid mode of evolution in a G + C-rich region of mammalian genomes

A sequence of 10,621 base-pairs from the alpha-like globin gene cluster of rabbit has been determined. It includes the sequence of gene zeta 1 (a pseudogene for the rabbit embryonic zeta-globin), the functional rabbit alpha-globin gene, and the theta 1 pseudogene, along with the sequences of eight C repeats (short interspersed repeats in rabbit) and a J sequence implicated in recombination. The region is quite G + C-rich (62%) and contains two CpG islands. As expected for a very G + C-rich region, it has an abundance of open reading frames, but few of the long open reading frames are associated with the coding regions of genes. Alignments between the sequences of the rabbit and human alpha-like globin gene clusters reveal matches primarily in the immediate vicinity of genes and CpG islands, while the intergenic regions of these gene clusters have many fewer matches than are seen between the beta-like globin gene clusters of these two species. Furthermore, the non-coding sequences in this portion of the rabbit alpha-like globin gene cluster are shorter than in human, indicating a strong tendency either for sequence contraction in the rabbit gene cluster or for expansion in the human gene cluster. Thus, the intergenic regions of the alpha-like globin gene clusters have evolved in a relatively fast mode since the mammalian radiation, but not exclusively by nucleotide substitution. Despite this rapid mode of evolution, some strong matches are found 5' to the start sites of the human and rabbit alpha genes, perhaps indicating conservation of a regulatory element. The rabbit J sequence is over 1000 base-pairs long; it contains a C repeat at its 5' end and an internal region of homology to the 3'-untranslated region of the alpha-globin gene. Part of the rabbit J sequence matches with sequences within the X homology block in human. Both of these regions have been implicated as hot-spots for recombination, hence the matching sequences are good candidates for such a function. All the interspersed repeats within both gene clusters are retroposon SINEs that appear to have inserted independently in the rabbit and human lineages.     

Systematic assessment of reduced representation bisulfite sequencing to human blood samples: A promising method for large-sample-scale epigenomic studies

Complementary to the time- and cost-intensive direct bisulfite sequencing, we applied reduced representation bisulfite sequencing (RRBS) to the human peripheral blood mononuclear cells (PBMC) from YH, the Asian individual whose genome and epigenome has been deciphered in the YH project and systematically assessed the genomic coverage, coverage depth and reproducibility of this technology as well as the concordance of DNA methylation levels measured by RRBS and direct bisulfite sequencing for the detected CpG sites. Our result suggests that RRBS can cover more than half of CpG islands and promoter regions with a good coverage depth and the proportion of the CpG sites covered by the biological replicates reaches 80-90%, indicating good reproducibility. Given a smaller data quantity, RRBS enjoys much better coverage depth than direct bisulfite sequencing and the concordance of DNA methylation levels between the two methods is high. It can be concluded that RRBS is a time and cost-effective sequencing method for unbiased DNA methylation profiling of CpG islands and promoter regions in a genome-wide scale and it is the method of choice to assay certain genomic regions for multiple samples in a rapid way.     

Genomic sequence analysis of a potential QTL region for fat trait on pig chromosome 6

On pig chromosome 6, the SW71 microsatellite is located in the region corresponding to several quantitative trait loci (QTL), such as those for intramuscular fat content and for body weight at 4 weeks of age. The genomic sequence of approximately 909 kb was obtained from seven BAC clones encompassing the SW71 region corresponding to human 18q11.21-q11.22. By searching the NCBI GenBank using BLASTX and BLASTN, this 909-kb segment was found to contain eight genes, RAB31, TXNDC2, VAPA, APCDD1, NAPG, FAM38B, C18orf30, and C18orf58, and one putative gene (DN119777). The average G + C content in the sequence of this contig was 45.75% and 33 CpG islands were detected. CpG islands were scattered throughout the region in which most of the putative genes were located. Dense CpG islands of approximately 840 bp were observed, including within the 5' UTR and exon 1 of the orthologs of the RAB31, VAPA, APCDD1, and NAPG genes. Comparative analysis of conserved segments of six species showed that K(a)/K(s) ratios of the TXNDC2 gene in collinear and rearranged segments were significantly different at 4.1 and 1.3, respectively. In conclusion, we demonstrated the genomic organization of pig chromosome 6, including the gene order surrounding SW71, which provides important information for comparative mapping. Moreover, the genes revealed in this study may be positional candidate genes associated with QTL on chromosome 6 that affect fat deposition in pigs.