A revision of the human XIST gene organization and structural comparison with mouse Xist

The XIST gene plays an essential role in X Chromosome (Chr) inactivation during the early development of female humans. It is believed that the XIST gene, not encoding a protein, functions as an RNA. The XIST cDNA is unusually long, as its full length is reported to be 16.5 kilobase pairs (kb). Here, comparison of sequences from the genomic interval downstream to the 3′ end of the human XIST gene against the human EST database brought to light a number of human EST sequences that are mapped to the region. Furthermore, PCR amplification of human cDNA libraries and RNA fluorescence in situ hybridization (RNA-FISH) demonstrate that the human XIST gene has additional 2.8 kb downstream sequences which have not been documented as a part of the gene. These data show that the full-length XIST cDNA is, in fact, 19.3 kb, not 16.5 kb as previously reported. The newly defined region contains an intron that may be alternatively spliced and seven polyadenylation signal sequences. Sequences in the newly defined region show overall sequence similarity with the 3′ terminal region of mouse Xist, and three subregions exhibit quite high sequence conservation. Interestingly, the new intron spans the first two subregions that are absent in one of the two isoforms of mouse Xist. Taken together, we revise the structure of human XIST cDNA and compare cDNA structures between human and mouse XIST/Xist. Received: 3 August 1999 / Accepted: 15 November 1999     

Cloning, Structure, and Chromosome Localization of the Mouse Glutaryl-CoA Dehydrogenase Gene

Glutaryl-CoA dehydrogenase (GCDH) is a nuclear-encoded, mitochondrial matrix enzyme. In humans, deficiency of GCDH leads to glutaric acidemia type I, an inherited disorder of amino acid metabolism characterized by a progressive neurodegenerative disease. In this report we describe the cloning and structure of the mouse GCDH (Gcdh) gene and cDNA and its chromosomal localization. The mouse Gcdh cDNA is 1.75 kb long and contains an open reading frame of 438 amino acids. The amino acid sequences of mouse, human, and pig GCDH are highly conserved. The mouse Gcdh gene contains 11 exons and spans 7 kb of genomic DNA. Gcdh was mapped by backcross analysis to mouse chromosome 8 within a region that is homologous to a region of human chromosome 19, where the human gene was previously mapped.     

Structure and chromosomal localization of the mouse oncomodulin gene

The rat gene encoding oncomodulin (OM), a small calcium-binding protein, is under the control of a solo LTR derived from an endogenous intracisternal A-particle. The latter sequence is the only OM promoter analyzed so far. In order to study cell-type-specific OM expression in a species lacking LTR sequences in the OM locus, we initially synthesized an OM cDNA from mouse placenta. By sequencing, we found a 137-bp-long 5 leader region that differed markedly from its rat counterpart but had high similarity to several mouse genomic sequences. Primers specific to this sequence in addition with primers specific for an exon 2/intron 2 sequence were used to screen a mouse ES cell line genomic P1 library. One positive clone contained the whole OM gene, including intron 1 of 25 kb and a 5 flanking region of 27 kb lacking an LTR. The region upstream of exon 1 contains no TATA or CCAAT boxes but has a homopurine/homopyrimidine stretch of 102 bp as well as a (CA)₂₂ repeat. The latter sequence is polymorphic and was therefore, used to map the OM gene to the distal end of the long arm of mouse Chromosome (Chr) 5 by interspecific backcross analysis. Additonally we localized the OM gene by in situ hybridization to the region G1-3 on Chr 5, confirming the genetic linkage results. Finally, the OM gene was found to be structurally conserved and to exist in a single copy in mammals.     

Molecular cloning and structural analysis of the cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) phosphoenolpyruvate carboxykinase (<Emphasis Type="Italic">CsPCK</Emphasis>) gene

Genomic sequence of the ATP-dependent phosphoeno/pyruvate carboxykinase (CsPCK) gene has been determined first from cucumber. Several putative clones were isolated in three rounds of genomic library screening with designated cDNA probes. These clones were analyzed via restriction digests, Southern hybridization, and nucleotide sequencing to ascertain the structure of theCsPCK gene. Analysis of a selected positive clone (λcscpk-4A) demonstrated that this gene consists of 13 exons and 12 introns, spanning 9 kb in the cucumber genome. Exon 1 contains only 23 nucleotides of the 5′-noncoding region of cucumberPCK cDNA, whereas Exon 2 comprises 12 nucleotides of the S′-noncoding region with an N-terminal PEPCK coding sequence. All the exon-intron junction sequences agree with the GT/AG consensus, except for the 5 donor site of Intron 7, where GC replaces the GT consensus. As with rice (Oryza sativa), cucumber contains only one copy of theCsPCK gene in its haploid genome. The overall number of exons and the structure of this gene are similar to those for bothArabidopsis Chromosome 4 (Atg4)PCK and the rice PCX genes, which contain 13 and 12 exons, respectively. Two additionalArabidopsis PCK genes can be found in the fifth chromosome (Atg5), which contains 9 exons and 8 introns (with 628 and 670 amino acids, respectively) of the PEPCK peptide. TheCsPCK gene promoter has conserved plant-specific as-acting elements within 2 kb of the 5’ flanking region. Several common cis-acting elements of the isocitrate lyase (icl) and malate synthase(ms) gene promoters, identified in theCsPCK gene, are responsible for the sugar response during plant development, especially at germination. These conserved elements are discussed here.     

Structure and expression of the Lyt-3 agene of C.AKR mice

The mouse Lyt-3 ^agene, which encodes the Lyt-3.1 T-cell surface alloantigen of the C.AKR strain, has been cloned, and the nucleotide sequence of its exons and more than 2 kb of 5 flanking sequence have been determined. The gene extends over approximately 16 kb of DNA and consists of six exons encoding leader, leader plus V-like domain, membrane-proximal, transmembrane, and cytoplasmic domains. The only difference between the coding region of the Lyt-3 ^agene and the cDNA sequences reported for Lyt-3 ^b(Nakauchi et al. 1987, Panaccio et al. 1987) is at position 77 of the mature protein where Lyt-3 ^aencodes serine and Lyt-3 ^bencodes arginine. This substitution must therefore be the basis for the serological distinction between the Lyt-3.1 and Lyt-3.2 alloantigens. Potential TATA and CAAT sequences, two Sp1 protein binding sites, two extended repeats of the dinucleotide, CA, a number of short inverted repeats, and an inverted segment of the mouse B1 repetitive sequence are found 5 to the Lyt-3 ^agene. Two consensus poly-A addition signals and a complete copy of the mouse B1 sequence are found 3 to the gene. Both B1-related regions are flanked by short direct repeats suggesting that they arose by an insertional mechanism. Cotransfection of the Lyt-3 ^agene together with a cloned Lyt-2 ^agene resulted in expression of both Lyt-2 and Lyt-3.1 on the surface of Ltk^– and BW5147 cells. Transfection of the Lyt-3 ^agene without Lyt-2 ^aled to expression of Lyt-3-related cellular RNA but did not result in surface expression of Lyt-3.1, suggesting that the Lyt-3 glycoprotein is not expressed on the cell surface in the absence of Lyt-2.     

Isolation and FISH mapping of Yeast Artificial Chromosomes (YACs) encompassing an allele of the Gm2 gene for gall midge resistance in rice

 Ten yeast artificial chromosomes (YACs) spanning the Gm2 locus have been isolated by screening high-density filters containing a total of approximately 7000 YAC (representing six genome equivalents) clones derived from a japonica rice, Nipponbare. The screening was done with five RFLP markers flanking a gall midge resistance gene, Gm2, which was previously mapped onto chromosome 4 of rice. This gene confers resistance to biotype 1 and 2 of gall midge (Orseolia oryzae), a major insect pest of rice in South and Southeast Asia. The RFLP markers RG214, RG329 and F8 hybridized with YAC Y2165. Two overlapping YAC clones (Y5212 and Y2165) were identified by Southern hybridization, with Gm2-flanking RFLP markers, and their inserts isolated. The purified YACs and RFLP markers flanking Gm2 were labeled and physically mapped by the fluorescence in situ hybridization (FISH) technique. All of them mapped to the long arm of chromosome 4 of the resistant variety of rice, ‘Phalguna’, confirming the previous RFLP mapping data. Received: 15 December 1997 / Accepted: 5 March 1998     

Syntenic conservation of HSP70 genes in cattle and humans

A phage library of bovine genomic DNA was screened for hybridization with a human HSP70 cDNA probe, and 21 positive plaques were identified and isolated. Restriction mapping and blot hybridization analysis of DNA from the recombinant plaques demonstrated that the cloned DNAs were derived from three different regions of the bovine genome. One region contains two tandemly arrayed HSP70 sequences, designated HSP70-1 and HSP70-2, separated by approximately 8 kb of DNA. Single HSP70 sequences, designated HSP70-3 and HSP70-4, were found in two other genomic regions. Locus-specific probes of unique flanking sequences from representative HSP70 clones were hybridized to restriction endonuclease-digested DNA from bovine-hamster and bovine-mouse somatic cell hybrid panels to determine the chromosomal location of the HSP70 sequences. The probe for the tandemly arrayed HSP70-1 and HSP70-2 sequences mapped to bovine chromosome 23, syntenic with glyoxalase 1, 21 steroid hydroxylase, and major histocompatibility class I loci. HSP70-3 sequences mapped to bovine chromosome 10, syntenic with nucleoside phosphorylase and murine osteosarcoma viral oncogene (v-fos), and HSP70-4 mapped to bovine syntenic group U6, syntenic with amylase 1 and phosphoglucomutase 1. On the basis of these data, we propose that bovine HSP70-1,2 are homologous to human HSPA1 and HSPA1L on chromosome 6p21.3, bovine HSP70-3 is the homolog of an unnamed human HSP70 gene on chromosome 14q22-q24, and bovine HSP70-4 is homologous to one of the human HSPA-6,-7 genes on chromosome 1.     

Ligation-mediated suppression-PCR as a powerful tool to analyse nuclear gene sequences in the green alga Chlamydomonas reinhardtii

To improve the analysis of unknown flanking DNA sequences adjacent to known sequences in nuclear genomes of photoautotrophic eukaryotic organisms, we established the technique of ligation-mediated suppression-PCR (LMS-PCR) in the green alga Chlamydomonas reinhardtii for (1) walking from a specific nuclear insertion fragment of random knockout mutants into the unknown flanking DNA sequence to identify and analyse disrupted genomic DNA regions and for (2) walking from highly conserved DNA regions derived from known gene iso-forms into flanking DNA sequences to identify new members of protein families. The feasibility of LMS-PCR for these applications was successfully demonstrated in two different approaches. The first resulted in the identification of a genomic DNA fragment flanking a nuclear insertion vector in a random knockout mutant whose phenotype was characterised by its inability to perform functional LHC state transitions. The second approach targeted the cab gene family. An oligonucleotide of a cabII gene, derived from a highly conserved region, was used to identify potential cab gene regions in the nuclear genome of Chlamydomonas. LMS-PCR combined with 3′ rapid amplification of cDNA ends (3′ RACE) and a PCR-based screening of a cDNA library resulted in the identification of the new cabII gene lhcb4. Both results clearly indicate that LMS-PCR is a powerful tool for the identification of flanking DNA sequences in the nuclear genome of Chlamydomonas reinhardtii. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cloning and characterization of a novel human TEKTIN1 gene

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. A new member of the tektin gene family was cloned from the human fetal brain cDNA library. We hence named it the human TEKTIN1 gene. TEKTIN1 cDNA consists of 1375 bp and has a putative open reading frame encoding 418 amino acids. The predicted protein is 48.3 kDa in size, and its amino acid sequence is 82% identical to that of the mouse, rat, and dog. One conserved peptide RPNVELCRD was observed at position number 323–331 of the amino acid sequence, which is a prominent feature of tektins and is likely to represent a functionally important protein domain. TEKTIN1 gene was mapped to the human chromosome 17 by BLAST search, and at least eight exons were found. Northern blot analysis indicated that TEKTIN1 was predominantly expressed in testis. By in-situ hybridization analysis, TEKTIN1 mRNA was localized to spermatocytes and round spermatids in the seminiferous tubules of the mouse testis, indicating that it may play a role in spermatogenesis.     

Cloning,expression and genomic structure of a novel human GNB2L1 gene,which encodes a receptor of activated protein kinase C (RACK)*

During a large-scale screen of a human fetal brain cDNA library, a novel human gene GNB2L1 encoding a novel RACK (receptor of activated protein kinase C) protein was isolated and sequenced. The cDNA is 1142 bp long and has a predicted open reading frame encoding 316 aa. The predicted protein shows higher similarity to rat RACK1 and many RACK proteins of different organisms including Drosophila, C. elegans, mouse, rat, human, C. fasciculata, zebrafish, A. thaliana, S. cerevisiae and so on, suggesting it is conserved during evolution. The gene was mapped to human chromosome 5q35.3, the telomer position of chromosome 5q, in which the disease gene for early-onset primary congenital lymphedema was mapped. Also, 5q35.3 is a frequently reported location for cytogenetic and molecular abnormalities in renal cell carcinomas. The gene has 8 exons and 7 introns. It is expressed ubiquitously in many human tissues detected by northern blot analysis and RT-PCR.     

Chromosomal assignment and expression pattern of the murine Lasp-1 gene

The human Lasp-1 (LIM and SH3 protein) gene was previously identified by differential screening of a breast cancer-derived metastatic lymph node cDNA library. It was located on the q12–q21 region of human chromosome 17 and was shown to be amplified and overexpressed in 12% of breast tumours. Lasp-1 defines a new LIM-protein subfamily, as it associates a C-terminal Src homology 3 (SH3) domain to a N-terminal LIM motif. In this study, the isolation and characterization of the cDNA encoding the mouse Lasp-1 protein are described, and it is shown to be highly conserved with its human counterpart. In addition to the LIM and SH3 domains, both human and mouse Lasp-1 contain an actin-binding domain. The mouse gene was mapped by in situ hybridization to the 11C–11D region of chromosome 11. Northern blot analysis shows that this gene is expressed from 7.5 to 17.5 days post-coitum of mouse embryogenesis and in almost all adult tissues.     

Cloning and mapping of bovine ZFX gene to the long arm of the X-chromosome (Xq34) and homologous mapping of ZFY gene to the distal region of the short arm of the bovine (Yp13), ovine (Yp12–p13), and caprine (Yp12–p13) Y Chromosome

A part of mouse Zfy-2 sequence was synthesized and used to screen a genomic library of the spinous country-rat (Tokudaia osimensis spp., 2n = 45). An isolated clone had the C-terminal region of Zfy, which consisted of 1190 bp, encoded 336 amino acid residues, and harbored 11 out of 13 zinc finger motifs. With this as a probe, a bovine testis cDNA library was screened. Two ZFX clones were isolated and their sequences combined. The short sequence, lacking part of the 5′ upstream region, was amplified by PCR or RT-PCR, cloned, and sequenced. A full-length ZFX was constructed by combining these three sequences. The bovine ZFX consisted of 5328 bp and encoded 800 amino acid residues, which contained 13 zinc finger motifs. ZFX was used as a probe for fluorescence in situ hybridization and was mapped to Xq34, different from its previously reported site at Xq21-q231. A SINE (short interspersed nuclear element) sequence consisting of 188 bp was found close to the end of the 3′-untranslated region of ZFX. The SINE sequence hybridized to all bovine chromosomes. ZFY is highly homologous with ZFX and, as a result, ZFY could be mapped simultaneously. ZFY was mapped to the distal region of the short arm of the Y Chromosome (Chr) (Yp13), contradicting the previously reported position Yq1. Ovine and caprine ZFY were also mapped with bovine ZFX. Both were mapped to the distal region of the short arm of the Y Chr (Yp12-p13). Ovine ZFX was mapped to a region close to the centromere of the X Chr (Xq13). Received: 23 July 1997 / Accepted: 30 September 1997     

Construction of a high-resolution genetic map and YAC-contigs in the tomato Tm-2a region

With the ultimate goal of cloning the Tobacco Mosaic Virus (TMV) resistance gene Tm-2a from tomato by means of positional cloning, a high-resolution map of a 4.3-cM region surrounding the Tm-2a gene has been constructed. In total, 13 RFLP and RAPD markers were mapped in close proximity to Tm-2a using 2112 individuals from an intraspecific Lycopersicon peruvianum backcross. The closest flanking markers were separated from Tm-2a by 0.05 cM on each side. Only one marker, the cDNA clone R12, co-segregated with Tm-2a. In order to physically cover the Tm-2a region, R12 and the flanking DNA marker TG207 were used to select homologous YAC clones. To-date, two YAC-contigs spanning approximately 340 kb and 360 kb have been constructed. The data obtained from these experiments indicate that recombination around the centromere of chromosome 9 is extremely suppressed.     

Molecular cloning of the human gene STK10 encoding lymphocyte-oriented kinase, and comparative chromosomal mapping of the human, mouse, and rat homologues

 LOK is a new and unique member of the STE20 family with serine/threonine kinase activity, and its expression is restricted mostly to lymphoid cells in mice. We cloned the cDNA encoding the human homologue of LOK. The amino acid sequence deduced from the cDNA shows a high similarity to that of mouse LOK, with 88% identity as a whole. The kinase domains at the N-terminus and the coiled-coil regions at the C-terminus are particularly conserved, showing 98% and 93% identity, respectively. Western blot analysis with mouse LOK-specific antibody detected 130 000 M _r LOK proteins in human and rat lymphoid cell lines and tissues. The gene encoding the LOK (STK10/Stk10) gene was mapped by fluorescence in situ hybridization to chromosome 5q35.1 in human, chromosome 11A4 in mouse, and chromosome 10q12.3 in rat. By virtue of polymorphic CA repeats found in the 3' untranslated region of the mouse Stk10 gene, the Stk10 locus was further pinpointed to chromosome 11 between D11Mit53 and D11Mit84, using the intersubspecific backcross mapping panel. These results established STK10 as a new marker of human chromosome 5 to define the syntenic boundary of human chromosomes 5 and 16 on mouse chromosome 11. Received: 28 September 1998 / Revised: 2 November 1998