Kb Of Genomic Sequence Research Articles

Engineering EGFP reporter constructs into a 200 kb human beta-globin BAC clone using GET Recombination

GET Recombination, a simple inducible homologous recombination system for Escherichia coli, was used to target insertion of an EGFP cassette between the start and termination codons of the beta-globin gene in a 200 kb BAC clone. The high degree of homology between the promoter regions of the beta- and delta-globin genes also allowed the simultaneous generation of a delta-globin reporter construct with the deletion of 8.8 kb of intervening sequences. Both constructs expressed EGFP after transient transfection of MEL cells. Similarly, targeting of the EGFP cassette between the promoter regions of the gamma-globin genes and the termination codon of the beta-globin gene enabled the generation of reporter constructs for both (A)gamma- and (G)gamma-globin genes, involving specific deletions of 24 and 29 kb of genomic sequence, respectively. Finally the EGFP cassette was also inserted between the epsilon- and beta-globin genes, with the simultaneous deletion of 44 kb of intervening sequence. The modified constructs were generated at high efficiency, illustrating the usefulness of GET Recombination to generate large deletions of specific sequences in BACs for functional studies. The establishment of stable erythropoietic cell lines with these globin constructs will facilitate the search for therapeutic agents that modify the expression of the individual globin genes in a physiologically relevant manner.

Mutations in the CNGB3 gene encoding the beta-subunit of the cone photoreceptor cGMP-gated channel are responsible for achromatopsia (ACHM3) linked to chromosome 8q21.

Achromatopsia is an autosomal recessive disorder featuring total colour blindness, photophobia, reduced visual acuity and nystagmus. While mutations in the CNGA3 gene on chromosome 2q11 are responsible for achromatopsia in a subset of patients, previous linkage studies have localized another achromatopsia locus, ACHM3, on chromosome 8q21. Using achromatopsia families in which CNGA3 mutations have been excluded, we refined the ACHM3 locus to a 3.7 cM region enclosed by markers D8S1838 and D8S273. Two yeast artificial chromosome (YAC) contigs covering nearly the entire ACHM3 interval were constructed. Database searches with YAC content sequences identified two overlapping high throughput genomic sequencing phase (HTGS) entries which contained sequences homologous to the murine cng6 gene encoding the putative beta-subunit of the cone photoreceptor cGMP-gated channel. Using RT-PCR and RACE, we identified and cloned the human cDNA homologue, designated CNGB3, which encodes an 809 amino acid polypeptide. Northern blot analysis revealed a major transcript of approximately 4.4 kb specifically expressed in the retina. The human CNGB3 gene consists of 18 exons distributed over approximately 200 kb of genomic sequence. Analysis of the CNGB3 gene in achromats revealed six different mutations including a missense mutation (S435F), two stop codon mutations (R203X and E336X), a 1 bp and an 8 bp deletion (1148delC and 819-826del) and a putative splice site mutation of intron 13. The 1148delC mutation was identified recurrently in several families, and in total was present on 11 of 22 disease chromosomes segregating in our families.

Isolation and Initial Characterization of a Novel Zinc Finger Gene, DNMT3L, on 21q22.3, Related to the Cytosine-5- Methyltransferase 3 Gene Family

We have isolated the DNMT3L gene that is related to the cytosine-5-methyltransferase 3 (DNMT3) family. The gene is located on chromosome 21q22.3 between the AIRE and the KIAA0653 genes and spans approximately 16 kb of genomic sequence. The encoded protein of 387 amino acids has a cysteine-rich region containing a novel-type zinc finger domain that is conserved in DNMT3A and DNMT3B but also in ATRX, a member of the SNF2 protein family. The novel domain, called an ADD (ATRX, DNMT3, DNMT3L)-type zinc finger, contains two subparts: a C2C2 and an imperfect PHD zinc finger. Expression of the DNMT3L mRNA was not detectable by Northern blotting; however, RT-PCR amplification revealed that it is expressed at low levels in several tissues including testis, ovary, and thymus.

Identification of 142 single nucleotide polymorphisms in 41 candidate genes for rheumatoid arthritis in the Japanese population.

Single-nucleotide polymorphisms (SNPs) can make an important contribution to our understanding of genetic backgrounds that may influence medical conditions and ethnic diversity. We undertook a systematic survey of genomic DNA for SNPs located not only in coding sequences but also in non-coding regions (e.g., introns and 5' flanking regions) of selected genes. Using DNA samples from 48 Japanese patients with rheumatoid arthritis (RA) as templates, we surveyed 41 genes that represent candidates for RA, screening a total of 104 kb of DNA (30 kb of coding sequences and 74 kb of non-coding DNA). Within this 104 kb of genomic sequences we identified 163 polymorphisms (1 per 638 bases on average), of which 142 were single-nucleotide substitutions and the remainder, insertions or deletions. Of the coding SNPs, 52% were non-synonymous substitutions, and non-conservative amino acid changes were observed in a quarter of those. Sixty-nine polymorphisms showed high frequencies for minor alleles (more than 15%) and 20 revealed low frequencies (<5%). Our results indicated a greater average distance between SNPs than others have reported, but this disparity may reflect the type of genes surveyed and/or the relative ethnic homogeneity of our test population.

Lhx9 and Lhx9α LIM-Homeodomain Factors: Genomic Structure, Expression Patterns, Chromosomal Localization, and Phylogenetic Analysis

Lhx9 is a LIM-homeodomain (LIM-hd) transcription factor expressed in the embryonic mouse brain. We report the isolation of Lhx9α, a cDNA encoding a truncated isoform of Lhx9 that lacks the recognition helix of the homeodomain and differs from Lhx9 cDNA in its 3′-coding and 3′-UTR sequences. Isolation of the Lhx9 gene showed that Lhx9 and Lhx9α are coded by six exons spanning 10 kb of genomic sequence and that Lhx9α is an isoform generated by alternative splicing of the fifth exon. Lhx9 was mapped to the subtelomeric region of chromosome 1. Further molecular analysis showed that Lhx9 is a new candidate gene for the unidentified dreher (dr) mutation in mouse. The comparison of genomic structure and molecular phylogenetic analysis led to the identification of six groups of LIM-hd proteins, a basis for further classification and knowledge of their evolutionary relationships. To investigate a possible role for Lhx9α, the expression patterns of Lhx9 and Lhx9α were compared during embryogenesis. Lhx9α was expressed at lower levels than Lhx9, with a similar but distinct pattern in the brain, especially in the neocortex. We suggest that Lhx9α could function as an endogenous dominant-negative form of Lhx9 during development, both to regulate in space and time the transcriptional effects of Lhx9 and to add a degree of refinement to the LIM-hd code.

Gene structure and chromosomal mapping of the rat smooth muscle calponin gene.

Smooth muscle cells (SMC) express a battery of lineage-restricted genes whose encoded proteins impart the unique contractile phenotype that characterizes this muscle type. While the encoded function of many SMC-restricted genes has been extensively analyzed, less is known about their position within the genome and the regulatory factors governing their transcription. In this report, we define the gene structure, 5' promoter analysis, and chromosomal mapping of the rat smooth muscle calponin (CnnI) gene. The rat CnnI gene is comprised of seven exons spanning approximately 8 kb of genomic sequence. The intron-exon boundaries of the rat CnnI gene match precisely those in human and mouse. Primer extension and RNase protection assays indicate two major transcription start positions (tsp). Comparative sequence analysis of the 5' promoter region reveals several conserved cis regulatory elements, including a TA-rich element within 30 nt of the tsp that could be a recognition site for TATA-binding protein and two CCAAT boxes. Transient and stable transfection studies support the hypothesis that distal regulatory elements confer SMC-restricted expression of CnnI. Finally, using an F2 intercross, we have mapped the rat CnnI gene to the telomeric end of Chromosome (Chr) 8. These studies provide additional information relating to the control of CnnI gene expression and provide a platform to begin assessing the potential linkage of CnnI to spontaneous and experimental disease phenotypes in rats.

Organization of a gene coding for an oviduct-specific glycoprotein (oviductin) in the hamster.

Hamster oviductin, a high molecular weight glycoprotein secreted by the oviducts, is believed to participate in fertilization and protection of the tubal epithelium. Expression of the oviductin gene is confined strictly to nonciliated secretory cells of the oviduct and is regulated by hormones. The objective of this study was to characterize the genomic organization and to identify potential regulatory elements implicated in the control of transcription of the oviductin gene. Polymerase chain reaction was performed on hamster genomic DNA, yielding 2.2 kb of the 5' flanking region as well as 13.6 kb of genomic sequence comprising the entire coding sequence of the oviductin gene distributed in 11 exons. Sequencing of the 5' flanking region revealed, among other elements, an almost perfect estrogen-responsive element (GGTCACTGTGACT), an atypical TATA box (TATTAA), and a perfect inverted Sp1 site located between the transcription start site and the atypical TATA box. Primer extension analyses indicated that the hamster oviductin transcript possesses an unusually short 5' untranslated region of only 14 nucleotides. The distinct organization of the hamster oviductin gene in the vicinity of the transcription start site provides an interesting ground for further functional studies.

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.

Genomic Organization, Mapping, Tissue Expression, and Hormonal Regulation of Trypsin-like Serine Protease (TLSP PRSS20), a New Member of the Human Kallikrein Gene Family

The cDNA for the trypsin-like serine protease gene (TLSP, HGMW-approved symbol PRSS20) has been recently identified. TLSP is expressed in brain and skin tissues but little else is known about this new serine protease gene. In this paper, we describe the complete genomic organization and precise mapping of the TLSP gene. This gene spans 5.3 kb of genomic sequence on chromosome 19q13.3–q13.4. The gene consists of six exons, the first of which is untranslated. All splice junctions follow the GT/AG rule, and the intron phases are identical to those of other kallikrein-like genes, including zyme (PRSS9), NES1 (PRSSL1), and neuropsin (PRSS19). Fine-mapping of the area indicates that TLSP lies downstream from the PSA, zyme, neuropsin, and NES1 genes. Significant sequence homologies were found between TLSP and other human kallikreins. Furthermore, there is conservation of the catalytic triad (histidine, aspartic acid, serine) and of the number of coding exons (five; the same in all members of the kallikrein gene family). We thus suggest that TLSP is a new member of the human kallikrein gene family. TLSP is expressed in many tissues including cerebellum, prostate, salivary glands, stomach, lung, thymus, small intestine, spleen, liver, and uterus. TLSP expression appears to be regulated by steroid hormones in the breast carcinoma cell line BT-474.

Homologous Genomic Fragments in the Mouse Pre-T Cell Receptor α (pTa) and Xist Loci

We recently characterized a genomic region located upstream of the mouse pre-T cell receptor α (pTa) gene, which controls pTa expression in pre-T cells. We now report an unexpected homology between this region and a region in the mouse X chromosome inactivation center between the 3′ end of the Xist gene and the start of an antisense transcript Tsix. The homology is extended over 4 kb of genomic sequence split by an expanded repeat region and is observed only in the mouse, not in the rat. Despite high sequence similarity to the pTa transcriptional enhancer, the homologous X chromosome fragment appears to have lost its enhancer activity. These data underscore the complex organization of the mouse genome and, in particular, of the X chromosome inactivation center.

Isolation and Characterization of a Novel Human paired-like Homeodomain-Containing Transcription Factor Gene, VSX1, Expressed in Ocular Tissues

Homeodomain transcription factors control cell fates during the development of all animals. The paired-like subfamily of homeodomain proteins has been particularly implicated in ocular development in different species. In this paper we report the cDNA sequence, genomic structure, localization, and expression data of a novel paired-like homeobox-containing gene, VSX1, isolated from a human embryonic craniofacial cDNA library using the degenerate-PCR approach. The composed VSX1 cDNA sequence of 1433 bp was predicted to encode a protein of 365 amino acid residues. Maximal homology at the protein level was identified with the paired-like homeoproteins of the CVC-domain family: 92–97% identity was seen in the homeodomain region with 55% overall identity to zebrafish and goldfish Vsx1 and 35% overall identity to goldfish Vsx2 and murine Chx10. The gene was found to consist of five exons that are distributed over 6.2 kb of genomic sequence. VSX1 was localized to the 20p11–q11 region, which is homologous with the distal part of mouse chromosome 2. Expression of VSX1 was detected in embryonic craniofacial and adult ocular tissues. Several ocular phenotypes have been mapped to the VSX1 region in both human and mouse genomes, and its candidacy for these disorders is discussed.

Sak kinase gene structure and transcriptional regulation

The Sak gene encodes a serine/threonine kinase, which is a member of the Polo family of mitotic regulators. Sak transcripts are present in S/G2/M phase cells, and in proliferating cell layers of the mouse embryo and adult tissues. In this report, we have characterized the murine Sak gene structure, the Sak chromosomal location, and identified the promoter. The murine Sak gene is located on the proximal arm of mouse chromosome 13, as determined by RFLP analysis. The murine gene comprises 15 coding exons spanning 16kb of genomic sequence, and encodes two alternately spliced transcripts. Sak-a, the predominant transcript, is encoded by 15 exons, while early termination of transcription and alternative splicing at exons 5 and 6 results in Sak-b. This truncated transcript encodes the complete kinase domain and a carboxyl end translated from 147bp of sequence contiguous with exon 5. Human Sak-a (Stk18) cDNA is reported to contain an insertion of sequence corresponding to the mouse Sak-b tail. Primer extension analysis of murine Sak revealed one major transcription start site at position -303bp relative to the start of translation. A genomic fragment of 3.5kb located 5' of the Sak transcriptional start drives expression of a luciferase-reporter gene in CHO and GC1-SPG cells in an orientation-dependent fashion. Using various Sak promoter/luciferase constructs, the core promoter region required for expression was located within 400bp of the message Cap site, and sequence further 5' strongly suppressed transcription.

Molecular Characterization of Zyme/Protease M/Neurosin (PRSS9), A Hormonally Regulated Kallikrein-like Serine Protease

The cDNA for the zyme/protease M/neurosin gene (HGMW-approved symbol PRSS9) has recently been identified. Zyme appears to play a role in Alzheimer disease as well as in breast cancer. In this paper, we describe the complete genomic organization of the zyme gene. Zyme spans 10.5 kb of genomic sequence on chromosome 19q13.3–q13.4. The gene consists of seven exons, the first two of which are untranslated. All splice junctions follow the GT/AG rule, and the intron phases are identical to those of many other genes belonging to the same family, i.e., the kallikreins, NES1, and neuropsin. Fine-mapping of the genomic locus indicates that zyme lies upstream of the NES1 gene and downstream from the PSA and KLK2 genes. Tissue expression studies indicate that zyme is expressed mainly in brain tissue, including spinal cord and cerebellum, in mammary gland, and in kidney and uterus. Zyme is regulated by steroid hormones in the breast carcinoma cell line BT-474. Estrogens and progestins, and to a lesser extent androgens, up-regulate the zyme gene in a dose-dependent manner.

Characterization of the human diacylglycerol kinase epsilon gene and its assessment as a candidate for inherited retinitis pigmentosa.

Human diacylglycerol kinase epsilon (hDGK epsilon) displays high selectivity for arachidonate-containing substrates and may be essential in the termination of signals transmitted through arachidonoyl-diacylglycerol and/or the synthesis of phospholipids with defined fatty acid composition. We herein report the genomic structure, chromosomal mapping, and mutation screening of hDGK epsilon gene. hDGK epsilon gene contains at least 12 exons spanning approximately 30 kb of genomic sequence and was mapped to chromosome 17q22 by fluorescence in situ hybridization. A search for disease gene linkage revealed that a locus for autosomal dominant retinitis pigmentosa (adRP) known as RP17 resided in that region, and Northern blot analysis showed that hDGK epsilon was expressed in human retina. The hDGK epsilon gene was then localized to one of the YAC clones containing a STS marker for the RP17 locus by YAC contig mapping. Direct sequencing following PCR amplification of two affected DNA samples from that type of adRP patients, however, did not reveal any mutation in hDGK epsilon exons.

Cloning of Fatso (Fto), a novel gene deleted by the Fused toes (Ft) mouse mutation.

The Fused toes (Ft) mouse mutation was created by insertional mutagenesis, resulting in the deletion of several hundred kb of genomic sequences of mouse Chromosome (Chr) 8. Mice heterozygous for the Ft mutation are characterized by partial syndactyly of forelimbs and massive thymic hyperplasia indicating that programmed cell death is affected. Homozygous Ft/Ft embryos die at midgestation and show severe malformations of craniofacial structures. Furthermore, establishment of left-right asymmetry is random. Here we report on the positional cloning of a novel gene by exon trap analysis of a genomic clone encoding wild-type sequences corresponding to parts of the deletion in Ft mutants. RT-PCR experiments demonstrated that the newly identified gene, Fatso (Fto), is expressed throughout embryonic development. Wide expression was also found in tissues of adult mice. We show that expression of Fto is completely absent in mouse embryonic fibroblasts homozygous for the Ft mutation. In addition, we isolated the full-length cDNA which encodes a putative 58-kDa protein showing no similarities to known proteins or protein motifs. The expression data of Fto define it as a candidate gene involved in processes such as programmed cell death, craniofacial development, and establishment of left-right asymmetry.

A random survey of the Cryptosporidium parvum genome.

Cryptosporidium parvum is an obligate intracellular pathogen responsible for widespread infections in humans and animals. The inability to obtain purified samples of this organism's various developmental stages has limited the understanding of the biochemical mechanisms important for C. parvum development or host-parasite interaction. To identify C. parvum genes independent of their developmental expression, a random sequence analysis of the 10.4-megabase genome of C. parvum was undertaken. Total genomic DNA was sheared by nebulization, and fragments between 800 and 1,500 bp were gel purified and cloned into a plasmid vector. A total of 442 clones were randomly selected and subjected to automated sequencing by using one or two primers flanking the cloning site. In this way, 654 genomic survey sequences (GSSs) were generated, corresponding to >320 kb of genomic sequence. These sequences were assembled into 408 contigs containing >250 kb of unique sequence, representing approximately 2.5% of the C. parvum genome. Comparison of the GSSs with sequences in the public DNA and protein databases revealed that 107 contigs (26%) displayed similarity to previously identified proteins and rRNA and tRNA genes. These included putative genes involved in the glycolytic pathway, DNA, RNA, and protein metabolism, and signal transduction pathways. The repetitive sequence elements identified included a telomere-like sequence containing hexamer repeats, 57 microsatellite-like elements composed of dinucleotide or trinucleotide repeats, and a direct repeat sequence. This study demonstrates that large-scale genomic sequencing is an efficient approach to analyze the organizational characteristics and information content of the C. parvum genome.

A bipartite sequence element associated with matrix/scaffold attachment regions.

We have identified a MAR/SAR recognition signature (MRS) which is common to a large group of matrix and scaffold attachment regions. The MRS is composed of two degenerate sequences (AATAAYAA and AWWRTAANNWWGNNNC) within close proximity. Analysis of >300 kb of genomic sequence from a variety of eukaryotic organisms shows that the MRS faithfully predicts 80% of MARs and SARs. In each case where we find a MRS, the corresponding DNA region binds specifically to the nuclear scaffold. Although all MRSs are associated with a SAR, not all known SARs and MARs contain a MRS, suggesting that at least two classes exist, one containing a MRS, the other not. Evidence is presented that the two sequence elements of the bipartite MRS occupy a position on the nucleosome near the dyad axis, together creating a putative protein binding site. The identification of a MAR- and SAR-associated DNA element is an important step forward towards understanding the molecular mechanisms of these elements. It will allow: (i) analysis of the genomic location of SARs, e.g. in relationship to genes, based on sequence information alone, rather than on the basis of an elaborate biochemical assay; (ii) identification and analysis of proteins that specifically bind to the MRS.

New gene family defined by MORC, a nuclear protein required for mouse spermatogenesis.

Mammalian spermatogenesis is a complex developmental process. The analysis of mouse mutations has provided insight into biochemical pathways required for completion of this process. We previously described the autosomal recessive mouse morc TgN(Tyr)1Az(microrchidia) mutation, a serendipitous transgenic insertional mutation which causes arrest of spermatogenesis prior to the pachytene stage of meiosis prophase I. We now report the molecular characterization of the morc locus and positional cloning of a gene disrupted by the morc TgN(Tyr)1Az mutation. This gene, which we term Morc, encodes a 108 kDa protein expressed specifically in male germ cells. The transgene integrated within the first intron of Morc and was accompanied by an intragenic deletion of approximately 13 kb of genomic sequences, removing exons 2-4 and abrogating expression of the wild-type transcript. Analysis of the MORC protein sequence revealed putative nuclear localization signals, two predicted coiled-coil structural motifs and limited homology to GHL (GyraseB, Hsp90, MutL) ATPase. Epitope-tagged MORC protein expressed in COS7 cells localized to the nucleus. We also cloned the human MORC homolog and show that it too is testis-specific, but closely related human genes are transcribed in multiple somatic tissues. Homologous proteins are also present in zebrafish, nematodes, slime mold and plants. Thus, cloning of Morc defines a novel gene family whose members are likely to serve important biological functions in both meiotic and mitotic cells of multicellular organisms.

Characterization of Novel Promoter and Enhancer Elements of the Mouse Homologue of the Dent Disease Gene, CLCN5, Implicated in X-Linked Hereditary Nephrolithiasis

The murine homologue of the human chloride channel gene, CLCN5, defects in which are responsible for Dent disease, has been cloned and characterized. We isolated the entire coding region of mouse Clcn5 cDNA and ∼45 kb of genomic sequence embracing the gene. To study its transcriptional control, the 5′ upstream sequences of the mouse Clcn5 gene were cloned into a luciferase reporter vector. Deletion analysis of 1.5 kb of the 5′ flanking sequence defined an active promoter region within 128 bp of the putative transcription start site, which is associated with a TATA motif but lacks a CAAT consensus. Within this sequence, there is a motif with homology to a purine-rich sequence responsible for the kidney-specific promoter activity of the rat CLC-K1 gene, another member of the chloride-channel gene family expressed in kidney. An enhancer element that confers a 10- to 20-fold increase in the promoter activity of the mouse Clcn5 gene was found within the first intron. The organization of the human CLCN5 and mouse Clcn5 gene structures is highly conserved, and the sequence of the murine protein is 98% similar to that of human, with its highest expression seen in the kidney. This study thus provides the first identification of the transcriptional control region of, and the basis for an understanding of the regulatory mechanism that controls, this kidney-specific, chloride-channel gene.

Characterization of loricrin regulation in vitro and in transgenic mice

We have previously shown that the promoter of a 6.5 kb mouse loricrin clone contains a functional AP-1 element and directs tissue-specific, but not differentiation-specific, expression. We now report the isolation of a 14-kb genomic clone containing an additional 7 kb of genomic sequence. The additional sequences limit expression of a reporter construct to differentiated keratinocytes in culture. The expression of the 6.5-kb and 14-kb loricrin constructs were also analyzed in transgenic mice. Significantly, loricrin was found in all layers of the epidermis of the 6.5-kb transgenics, including basal and spinous cells. The expression of the 14-kb clone was indistinguishable from that of the endogenous gene, confirming that the additional sequences contain negative regulatory elements that restrict loricrin expression to the granular layer in vivo. In addition, we show the AP-1 element localized in the loricrin proximal promoter is necessary but not sufficient for expression of the loricrin gene in vivo in transgenic mice. Finally, to gain further insight into how AP-1 family members regulate expression of the loricrin gene, we co-transfected the loricrin reporter constructs with expression plasmids for various fos and jun family members and demonstrated that c-Fos/Jun-B heterodimers could mimic the differentiation-specific induction of loricrin.