Cloning, mapping and characterization of the human RAB27A gene

Choroideremia (CHM) is an X-linked retinal degenerative disease that results from mutations in Rab Escort Protein-1 (REP1). REP1 acts in the prenylation of Rab GTPases, regulators of intracellular protein trafficking. Rab27a is unique among Rabs in that it is selectively unprenylated in CHM cells, suggesting that the degenerative process in CHM may result from unprenylation and consequent loss-of-function of Rab27a. As a first step towards the analysis of the Rab27a protein in patients, we report here the characterization of the human RAB27A gene. The putative protein encoded by this gene shares 96% identity with the previously cloned rat homologue. The RAB27A gene comprises five coding exons and two non-coding exons, of which one is alternatively used, and spans approximately 65 kb of DNA. There are three alternative poly-A addition sites in the long 3' UTR and also six potential single-nucleotide polymorphisms. The gene is located on chromosome 15q15-21.1, as determined by fluorescent in situ hybridization, and between markers D15S209 and AFM321ZD5 by radiation hybrid mapping.     

Cloning,characterization and chromosome mapping of the human SMAP1 gene

Stromal membrane associated protein (smap-1) is a new murine cell surface molecule on the stromal cells. The murine smap-1 protein is induced in stromal cells by the contact with erythroid cells, which suggests that this protein may be involved in the haematopoietic progenitor cells to stromal cells interactions.Here we report the structure, map location and expression analysis of the human SMAP1 gene, which cover approximately 100 kb on chromosome 6 between D6S455 and D6S1673 markers. This gene is composed of 11 exons and encodes a 468-amino-acid protein, which shows an 86% of homology with the murine smap-1 protein. The expression of smap-1 in erythropoietic organs as well as the correlation with the erythropoietic activity of the haematopoietic organs suggest that smap-1 is induced in stromal cells by the contact with erythroid cells, defining smap-1 as a key molecule that induced an erythropoietic microenvironment in haematopoietic organs. The high sequence conservation between murine and human SMAP1, as well as its expression in bone marrow, strongly suggest conserved functions of this protein in both organisms. Recently, a constitutional translocation t(6;10)(q13;q22) has been described in a patient with severe aplastic anaemia. SMAP1 gene localizes to 6q13 and is probably implicated in erythropoiesis, therefore it remains as an interesting candidate gene.     

Cloning, characterization, and mapping of the gene encoding the human G protein gamma 2 subunit

G proteins play vital roles in cellular responses to external signals. The specificity of G protein-receptor interaction is mediated mostly by the gamma-subunit and the individual members of the gamma-subunit multigene family would hence be expected to each have a particular expression profile. In an experiment designed to isolate genes expressed predominantly in human testis we identified a cDNA fragment corresponding to the gamma2 gene. Although the protein sequence of the gamma2 subunit has previously been published, the cDNA sequence, expression pattern, genomic structure, and localisation of the human GNG2 gene have not been described. We report the complete sequence of the GNG2 cDNA which is 1066 bp long and contains an open reading frame encoding a protein of 71 amino acids. This protein is 100% homologous to the bovine, mouse, and rat G protein gamma2 subunit. The gene structure is very similar to that of other Ggamma-subunit genes in that there are two introns, one located in the 5' UTR and the other within the ORF. We show that this gene is expressed in a range of foetal tissues as well as adult testis, adrenal gland, brain, white blood cells and lung but not in adult liver, muscle, sperm, prostate gland nor in the testes of two different infertile patients. There is evidence that GNG2 is expressed in malignant tissues. Using two independent methods, we have mapped the human GNG2 gene to chromosome 14q21.     

Cloning, chromosome mapping and functional characterization of a human homologue of murine gtse-1 (B99) gene

Murine Gtse-1 (G(2) and S phase expressed protein), previously named B99, is a wt-p53 inducible gene that encodes a microtubule-localized protein which is able to induce G(2)/M phase accumulation when ectopically expressed. Here we report the cloning and characterization of a new cDNA (GTSE-1) encoding a human homologue of the mouse Gtse-1 protein. Chromosome mapping of mouse and human genes assigned Gtse-1 to chromosome 15 and GTSE-1 to chromosome 22q13.2-q13.3 in a region with conserved synteny to that where Gtse-1 mapped. Analysis of the genomic structure revealed that GTSE-1 contains at least 11 exons and 10 introns, spanning approximately 33kb of genomic DNA. Similar to murine Gtse-1, the product of GTSE-1 localized to the microtubules, was able to delay G(2)/M progression when ectopically expressed and was cell cycle regulated. Taken together, these results indicate GTSE-1 as the human functional homologue of murine Gtse-1.     

Molecular cloning, mapping and characterization of the human neurocalcin delta gene (NCALD)

We identified a new human gene that encodes a cognate of the bovine neurocalcin delta from a human fetal brain cDNA library; hence we named it human neurocalcin delta (NCALD) gene. The deduced polypeptide product of the cDNA is 22 kDa in size, and its amino acid sequence is 100% and 99% identical to that of the bovine and chicken neurocalcin, respectively. Northern blots showed that the NCALD gene is more abundantly expressed in brain, testis, ovary and small intestine. Tissue in situ hybridization confirmed the existence of the NCALD mRNA in the adult human testis. Radiation hybrid panel mapping localized the gene to chromosome 8 between molecular markers D8S270 and D8S257.     

Cloning and characterization of alpha(s2)-casein gene of Riverine buffalo.

The present study was carried out to characterize the alpha(s2)-casein gene in Riverine buffalo. Total RNA was extracted from the mammary tissue of buffalo and alpha(s2)-casein cDNA were synthesized by RT-PCR, then cloned using pDRIVE-cloning vector and sequenced. The sequencing revealed that the size of alpha(s2)-casein was 669 bp with GC content of 41.11%. The gene encoded for 222 amino acid precursors and that it possessed 15 amino acids signal peptide. The similarity of buffalo alpha(s2)-casein mRNA sequence with that of cattle, sheep, goat, pig and camel were estimated as 97.9, 93.6, 93.4, 73.5 and 73.0%, respectively. In the phylogenetic trees, constructed from the data of the alpha(s2)-casein mRNA sequences as well as protein sequences, it has been observed that the cattle and buffalo were in the same group whereas sheep and goat formed another group. The camel and swine were placed in two separate groups.     

Isolation and partial characterization of the structural gene for human acid alpha glucosidase.

Genetic deficiency of acid alpha glucosidase (GAA) results in glycogen storage disease type II. To study the disease at the molecular level, we have previously isolated and sequenced the cDNA (3.6 kb) for human GAA. We have now isolated the structural gene, mapped and determined the position and size of the exons containing the entire cDNA, and determined the sequence of the intron-exon junctions. The structural gene is approximately 28 kb and contains 20 exons. The first exon has only 5' untranslated sequence and is separated by an approximately 2.7-kb intron from the second exon that contains the initiation ATG. The second as well as the last exon are quite large (578 and 607 bp) with the remainder of the exons ranging from 85-187 bp. Additionally, two new restriction fragment length (RFLPs) for Xba I and Stu I are described at the GAA locus, one of which is most 5' of the eight RFLPs we have previously described.     

Cloning, expression, and physical mapping of the 3beta-hydroxysteroid dehydrogenase gene cluster (HSD3BP1-HSD3BP5) in human.

Seven members of the human 3beta-hydroxysteroid dehydrogenase (3beta-HSD) gene family (HGMW-approved symbols HSD3BP1-HSD3BP5) have been cloned and physically mapped. HSD3B1 and 2 express 3beta-HSD enzymes; HSD3Bpsi1-5 are unprocessed pseudogenes that are closely related to HSD3B1 and 2 but contain no corresponding open reading frames. mRNA is expressed from psi4 and psi5 in several tissues, but with altered splice sites that disrupt reading frames. A 0.5-Mb contig of 3 yeast artificial chromosome and 32 bacterial artificial chromosome genomic clones contained no additional members of the gene family. The seven genes and pseudogenes mapped within 230 kb in the order HSD3Bpsi5-psi4-psi3-HSD3B1-psi1-psi2 -HSD3B2. HSD3B1 and 2 are in direct repeat, 100 kb apart. Six HSD3B2 mutations involve substitutions that are present in several of the pseudogenes. In four cases, mutations arose in CpG sites that are conserved within the gene cluster. The tendency for CpG sites to mutate by transition provides an adequate explanation for these HSD3B2 mutations, which are unlikely to be due to recombination or conversion within the gene family.     

Cloning and characterization of the human beta-glucuronidase gene

We have isolated a cosmid clone that contains GUSB, the human gene encoding beta-glucuronidase. The 21-kb gene contains 12 exons ranging from 85 to 376 bp in length. Exon 6 corresponds to the 153-bp deletion in the shorter of two types of cDNAs reported earlier, supporting the hypothesis that this cDNA arose by alternate splicing leading to exon skipping. The insert contains 4.2 kb of sequence upstream from the first exon and 6 kb 3' of the last exon. The clone expresses human beta-glucuronidase in stably transformed rat XCtk- cells. Comparison of the human gene organization with that recently reported for the murine beta-glucuronidase gene revealed that the intron/exon boundaries are identical. In the splice junctions, the most highly conserved regions are those identified as consensus sequences, and these are at least as highly conserved as bases encoding the translated portion of the gene.