Molecular Cloning and Chromosomal Localization to 17q21 of the Human WNT3 Gene

In mouse mammary tumors, the Wnt-3 gene can be activated by proviral insertion. Here we report on the isolation of a human homolog, WNT3. A genomic clone was isolated by use of mouse Wnt-3 sequences as a probe, after which cDNA containing most of the protein-encoding domain of the human gene was obtained by PCR. Comparison between the deduced mouse and human WNT-3 protein sequences showed four changes in 333 amino acids. WNT3 is located on chromosome 17q21. The gene was not found to be amplified or rearranged in a collection of human breast tumors.     

Cloning of the Human Monocarboxylate Transporter MCT3 Gene: Localization to Chromosome 22q12.3–q13.2

Lactate transport across cell membranes is mediated by a family of proton-coupled monocarboxylate transporters (MCTs). The retinal pigment epithelium (RPE) expresses a unique member of this family, MCT3. A portion of the human MCT3 gene was cloned by polymerase chain reaction using primers designed from rat RPE MCT3 cDNA sequence. The human genomic sequence was used to design primers to clone human MCT3 cDNA and to identify a bacterial artificial chromosome clone containing the human MCT3 gene. The human MCT3 cDNA contained a 1512-nucleotide open reading frame with a deduced amino sequence 85% identical to rat MCT3. Comparison of the cDNA and genomic sequences revealed that the MCT3 gene was composed of five exons distributed over 5 kb of DNA. The exon–intron borders were conserved between the human and the chicken MCT3 genes. Using radiation hybrid mapping, the MCT3 gene was mapped to chromosome 22 between markers WI11639 and SGC30687. A search of chromosome 22 in the Sanger Centre database confirmed the location of the human MCT3 gene at 22q12.3–q13.2.     

Localization and Recycling of gp27 (hp24γ3): Complex Formation with Other p24 Family Members

We report here the characterization of gp27 (hp24gamma3), a glycoprotein of the p24 family of small and abundant transmembrane proteins of the secretory pathway. Immunoelectron and confocal scanning microscopy show that at steady state, gp27 localizes to the cis side of the Golgi apparatus. In addition, some gp27 was detected in COPI- and COPII-coated structures throughout the cytoplasm. This indicated cycling that was confirmed in three ways. First, 15 degrees C temperature treatment resulted in accumulation of gp27 in pre-Golgi structures colocalizing with anterograde cargo. Second, treatment with brefeldin A caused gp27 to relocate into peripheral structures positive for both KDEL receptor and COPII. Third, microinjection of a dominant negative mutant of Sar1p trapped gp27 in the endoplasmic reticulum (ER) by blocking ER export. Together, this shows that gp27 cycles extensively in the early secretory pathway. Immunoprecipitation and coexpression studies further revealed that a significant fraction of gp27 existed in a hetero-oligomeric complex. Three members of the p24 family, GMP25 (hp24alpha2), p24 (hp24beta1), and p23 (hp24delta1), coprecipitated in what appeared to be stochiometric amounts. This heterocomplex was specific. Immunoprecipitation of p26 (hp24gamma4) failed to coprecipitate GMP25, p24, or p23. Also, very little p26 was found coprecipitating with gp27. A functional requirement for complex formation was suggested at the level of ER export. Transiently expressed gp27 failed to leave the ER unless other p24 family proteins were coexpressed. Comparison of attached oligosaccharides showed that gp27 and GMP25 recycled differentially. Only a very minor portion of GMP25 displayed complex oligosaccharides. In contrast, all of gp27 showed modifications by medial and trans enzymes at steady state. We conclude from these data that a portion of gp27 exists as hetero-oligomeric complexes with GMP25, p24, and p23 and that these complexes are in dynamic equilibrium with individual p24 proteins to allow for differential recycling and distributions.     

拟南芥Rab1家族基因的克隆和功能研究

Yptl蛋白是酵母唯一的Rab1 GTP酶,调控囊泡从内质网到高尔基体的运输.酵母温敏突变株 ASY01是一个Ypt1基因功能部分缺失菌株,在26℃可以正常生长,但在37℃不能生长.拟南芥有4个Rab1基因,分别是AtRab1A1、AtRab1B1、AtRab1B2、AtRab1C1.克隆了所有4个AtRab1基因,构建酵母表达载体,转化温敏突变型酵母ASY01.温度敏感性实验结果表明,所有转基因菌株在37℃都恢复正常生长.说明拟南芥4个Rab1基因都与酵母Ypt1基因功能互补,都具有调节囊泡从内质网到高尔基体运输的功能.     

人Xp11.2区4.3MbYAC重叠群：大尺度限制图与CpG岛分析

人Ｘｐ１１．２区域具有重要的医学遗传学和基础遗传学价值,它包含很多遗传疾病基因,且至少包含一个逃避Ｘ染色体失活的位点,非常规的基化多态也有发现。我们利用这一区域已知的一系列ＤＮＡ位标,从我们构建的ＹＡＣ库中筛选出一系列ＹＡＣ克隆。     

Quick Identification and Localization of CpG Islands in Large Genomic Fragments by Partial Digestion with Hpa II and Hha I

More than 50% of mammalian genes are associated with CpG islandsand thus they serve as a good gene marker. We have devised asimple method to scan large pieces of native or cloned genomicDNA for CpG islands. The method is based on the presence ofmultiple Hpa II and Hha I sites in CpG islands, at a frequency30 times higher than in the rest of the genome. The steps includecomplete digestion of DNA with a rare-cutting restriction endonuclease(to produce large fragments with defined ends), partial digestionwith Hpa II and Hha I, and subsequent Southern hybridizationwith an end probe. This identifies a CpG island as a clusterof sub-bands and, based on their electrophoretic mobility, onecan immediately locate the island relative to the ends. Formany vectors, universal probes flanking the cloning site areavailable, enabling the simultaneous analysis of a large numberof samples. We demonstrated the usefulness of the method byanalyzing known CpG islands in native genomic DNA and lambda,cosmid and P1 clones, and by isolating two novel transcribedislands from anonymous cosmid clones. Our method is quick, inexpensive,and can detect CpG islands with few or even no rare-cutter sites.     

Cloning and Gene Mapping of the Chromosome 13q14 Region Deleted in Chronic Lymphocytic Leukemia

Frequent deletions and loss of heterozygosity in a segment of chromosome 13 (13q14) in cases of B-cell chronic lymphocytic leukemia (CLL) have suggested that this malignancy is caused by inactivation of an unknown tumor suppressor gene located in this region. Toward the identification of the putative CLL tumor suppressor, we have constructed a high-resolution physical map of YAC, PAC, and cosmid contigs covering 600 kb of the 13q14 genomic region. In addition to densely positioned genetic markers and STSs, this map was further annotated by localization of 32 transcribed sequences (ESTs) using a combination of exon trapping, direct cDNA selection, sample sequencing of cosmids and PACs, and homology searches. On the basis of these mapping data, allelic loss analyses at 13q14 using CLL tumor samples allowed narrowing of the genomic segment encompassing the putative CLL gene to <300 kb. Twenty-three ESTs located within this minimally deleted region are candidate exons for the CLL-associated tumor suppressor gene.     

Identification of Members of the Dimocarpus Longan Flowering Locus T Gene Family with Divergent Functions in Flowering

Dimocarpus longan is a subtropical fruit crop whose year-round production relies on the application of KClO₃ to induce flowering; however, the mechanism by which this chemical causes flowering is yet unknown. To further characterize floral signaling in this species, we have isolated three longan FLOWERING LOCUS T (FT)-like genes and studied their activities by heterologous expression in Arabidopsis. Expression of two of these genes (DlFT2 and DlFT3) accelerates flowering, whereas expression of the third gene (DlFT1) causes delayed flowering and produced floral morphology defects. This anti-florigenic protein may be a member of a class of FT-like family involved in flowering time control in biennial and perennial species. Surprisingly, KClO₃ treatment also suppressed the expression of both DlFT2 and DlFT3 in a field trial.     

Gene Mapping of Usher Syndrome Type IIa: Localization of the Gene to a 2.1-cM Segment on Chromosome 1q41

Usher syndrome type II is associated with hearing loss and retinitis pigmentosa but not with any vestibular problems. It is known to be genetically heterogeneous, and one locus (termed USH2A) has been linked to chromosome 1q41. In an effort to refine the localization of USH2A, the genetic map of the region between and adjacent to the marker loci previously recognized as flanking USH2A (D1S70 and PPOL) is updated. Analysis of marker data on 68 Usher II families places the USH2A gene into a 2.1-cM region between the markers D1S237 and D1S229. The gene for transforming growth factor β2 (TGFB2) and the gene for the homeodomain box (HLX1) are both eliminated as candidates for USH2A, by virtue of their localization outside these flanking markers. The earlier finding of genetic heterogeneity was confirmed in six new families, and the proportion of unlinked Usher II families is estimated at 12.5%. The placement of the USH2A gene into this region will aid in the physical mapping and isolation of the gene itself.     

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.     

Chromosomal Mapping of Two Members of the Human Dynein Gene Family to Chromosome Regions 7p15 and 11q13 near the Deafness Loci DFNA 5 and DFNA 11

We mapped expressed tagged sequences (ESTs) corresponding to two human dynein heavy chain genes: β heavy chain of the outer dynein arm and heavy chain isotype 1B (DYH1B), by using somatic cell hybrids and radiation hybrid panels. The EST for the β heavy chain of the outer dynein arm mapped to chromosome region 7p15, and the EST for DYH1B mapped to 11q13.5. Two loci for nonsyndromic forms of deafness, DFNA5 and DFNA11, have previously been mapped to these two chromosomal regions. Including the gene for the axonemal light chain, hp28, we have mapped three different dynein genes near loci for different forms of nonsyndromic deafness. The hypothesis that mutations in some dynein genes are associated with nonsyndromic deafness should now be tested.     

Complete Structure of the Human Gc Gene: Differences and Similarities between Members of the Albumin Gene Family

The sequence of the human Gc gene, including 4228 base pairs of the 5′-flanking region and 8514 base pairs of the 3′ flanking region (55,136 in total), was determined from five overlapping λ phage clones. The sequence spans 42,394 base pairs from the cap site to the polyadenylation site, and it reveals that the gene is composed of 13 exons, which are symmetrically placed within the three domains of the Gc protein. The first exon is partially untranslated, as is exon 12, which contains the termination codon TAG. Exon 13 is entirely untranslated, but contains the polyadenylation signal AATAAA. Ten central introns split the coding sequence between codon positions 2 and 3 and between codon positions 3 and 1 in an alternating pattern, exactly as has been observed in the structure of the albumin and α-fetoprotein genes. The Gc gene has several distinctive features which set it apart from the other members of the family. First, the gene is smaller by two exons, which results in a protein some 130 amino acids shorter than albumin or AFP. This decrease in size may result from the loss of two internal exons during the evolutionary history of the Gc gene. Second, exons 6, 8, 9, and 11 are smaller than their counterparts in albumin or AFP by a total of 8 codons (1, 4, 1, and 2, respectively). Although the mRNA and protein expressed from the Gc gene are significantly smaller, the gene itself is about 2.5 times larger than the other genes of the family. There are 13 interspersed DNA repeats within the human Gc gene which are absent from the same positions in the albumin or AFP genes, and hence must have been inserted after the triplication event(s) that gave rise to the gene family. Despite the differences, the Gc gene is nonetheless recognizable as a member of the albumin family.     

Identification of an Interstitial 18p11.32-p11.31 Duplication Including the EMILIN2 Gene in a Family with Porokeratosis of Mibelli

Porokeratosis is a rare disease of epidermal keratinization characterized by the histopathological feature of the cornoid lamella, a column of tightly fitted parakeratocytic cells, whose etiology is still unclear. Porokeratosis of Mibelli is a subtype of porokeratosis presenting a single plaque or a small number of plaques of variable size located unilaterally on limbs. It frequently appears in childhood and occurs with a higher incidence in males. Cytogenetic analyses were performed in all members of the family on lesioned and uninvolved skin. An array-CGH analysis was also performed utilizing the Human Genome CGH Microarray Kit G3 400 with 5.3 KB overall median probe spacing. Gene expression was performed on skin fibroblasts. In this study, we describe a Caucasian healthy 4-year-old child and his father showing features of porokeratosis of Mibelli. Array-CGH analysis revealed an interstitial 429.5 Kb duplication of chromosome 18p11.32-p11.3 containing four genes, namely: SMCHD1, EMILIN2, LPIN2, and MYOM1 both in patient and his father. EMILIN2 resulted overexpressed on skin fibroblasts. Also other members of this family, without evident signs of porokeratosis, carried the same duplication. Among these genes, we focused our attention on elastin microfibril interfacer 2 (EMILIN2) gene. Apoptosis plays a fundamental role in maintaining epidermal homeostasis, balancing keratinocytes proliferation, and forming the stratum corneum. EMILIN2 is known to trigger the apoptosis of different cell lines negatively affecting cell survival. It is expressed in the skin. We could speculate that the duplication and overexpression of EMILIN2 cause an abnormal apoptosis of epidermal keratinocytes and alter the process of keratinization, even if other epigenetic and genetic factors could also be involved. Our results could contribute to a better understanding of the pathogenesis of porokeratosis of Mibelli.