Precise in situ localization of NCAM, ETS1, and D11S29 on human meiotic chromosomes

In order to sublocalize NCAM, ETS1, and the anonymous DNA fragment D11S29 within 11q23, in situ hybridization was performed on pachytene bivalents. Analysis of the grain distribution within the band 11q23 indicated that the chromosomal sublocalization of both NCAM and D11S29 was in 11q23.1, whereas ETS1 was found to be localized in 11q23.3. These results clearly demonstrate the usefulness of in situ hybridization applied to pachytene bivalents to obtain accurate gene sublocalization.     

Localization of the human NCAM gene to band q23 of chromosome 11: the third gene coding for a cell interaction molecule mapped to the distal portion of the long arm of chromosome 11

cDNA clones containing sequences coding for the murine neural cell adhesion molecule (N-CAM) were used in Southern hybridizations on human genomic DNA and demonstrated approximately 90% homology between human and murine NCAM genes. In situ hybridization with one of these clones was performed on human metaphase chromosomes and allowed the localization of the human NCAM gene to band q23 of chromosome 11. The genes for two other cell surface molecules believed to be involved in cell-cell interactions, Thy-1 and the delta chain of the T3-T cell receptor complex, have recently been localized to the same region of chromosome 11 in man. Moreover, this region of the human chromosome 11 appears to be syntenic to a region of murine chromosome 9 that also contains the staggerer locus: staggerer mice show abnormal neurological features which may be related to abnormalities in the conversion of the embryonic to the adult forms of the N-CAM molecule.     

Mapping studies and expression of genes located on human chromosome 11, band q23

Chromosome translocations and deletions may alter cellular proto-oncogenes and result in cellular changes that are important in the pathogenesis of malignancy. The region 11q23 is frequently involved in human malignancy. Utilizing a leukemic cell line with a reciprocal translocation involving 11q23 and somatic cell hybrids derived from this cell line, we analyzed five genes assigned to 11q23: NCAM, CD3D, CD3E, THY1, and ETS1. Our data showed no evidence of direct involvement of these genes in this leukemia but enabled a partial genetic map of this important region of the human genome to be constructed: 11cen--NCAM--CD3([G, D], E)--parallel--(ETS1, THY1)--11qter.     

Nine novel germline mutations of STK11 in ten families with Peutz-Jeghers syndrome

Peutz-Jeghers Syndrome (PJS) is an autosomal dominant hereditary disease characterized by hamartomatous polyposis involving the entire bowel. Recently STK11, a gene bearing a mutation responsible for PJS, was isolated. We investigated the entire coding region of STK11 in 15 unrelated PJS families by the PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) method and PCR-direct sequence analysis, and found nine different, novel mutations among ten of those families. One nonsense mutation and five different frameshift mutations (two families carried the same mutation), all of which would cause truncation of the gene product, were found in seven families; mutations found in five families were clustered within exon 6. Among these five mutations, three occurred at the mononucleotide-repeat region (CCCCCC) of codons 279–281, suggesting that this region is likely to be a mutational hotspot of this gene. One of the remaining three families carried a 3-bp in-frame deletion that would eliminate an asparagine residue within a kinase domain of the product; the other two carried intronic mutations at or adjacent to the consensus dinucleotide sequences of splice-acceptor or -donor sites, which were likely to lead to aberrant splicing. Received: 9 March 1998 / Accepted: 1 May 1998     

Fine mapping of the chromosome 11q22-23 region using PFGE, linkage and haplotype analysis; localization of the gene for ataxia telangiectasia to a 5cM region flanked by NCAM/DRD2 and STMY/CJ52.75, phi 2.22.

Using pulsed-field gel electrophoresis, and a range of different enzyme digests, we have established that both markers of each of the pairs CJ52.208/YNB3.12, NCAM/DRD2, and STMY/CJ52.75, on chromosome 11q22-23, show physical linkage on a single DNA fragment. We have also shown, using genetic linkage and haplotype analyses, that these markers lie within a region of approximately 18cM, which, it has been shown previously, is likely to contain the A-T gene. The relative positions of these marker loci, and the distance between them was determined in order to construct a detailed map which has allowed a more precise localization of the A-T gene. We have shown that in pairwise linkage analysis the strongest support for linkage to the A-T gene was with the STMY/CJ52.75 locus (Z = 5.59, theta = 0.0). A three-point analysis using the results from STMY/CJ52.75 and the closely linked marker phi 2.22 gave Z = 5.55, theta = 0.03. Despite persisting evidence of some linkage to Thy-1 our results are consistent with the existence of a single A-T locus on chromosome 11q22-23 and our best estimate of the position of this locus places it between NCAM/DRD2 and (STMY/CJ52.75, F2.22) (Z = 6.74), a region of approximately 5cM in males.     

Comparative mapping of the constitutional and tumor-associated 11;22 translocations.

The reciprocal t(11;22)(q23;q11) is the most common non-Robertsonian constitutional translocation in humans. The tumor-associated 11;22 rearrangement of Ewing sarcoma (ES) and peripheral neuroepithelioma (NE) is cytologically very similar to the 11;22 constitutional rearrangement. Using immunoglobulin light-chain constant region, ETS1 probes, and the technique of in situ hybridization, we previously were able to show that the constitutional and ES/NE breakpoints are different. As a first step toward isolating these translocation junctions and to further distinguish between them, we have made somatic cell hybrids. Cells from a constitutional 46,XX,inv(9),t(11;22) carrier and from an ES cell line with a t(11;22) were separately fused to a hypoxanthine-guanine phosphoribosyltransferase-deficient Chinese hamster cell line (RJK88). Resulting clones were screened with G-banding and Southern hybridization. Hybrid clones derived from the constitutional t(11;22) were established which contained the der(22) and both the der(22) and the der(11). Hybrid clones derived from the ES cell line containing the der(11) were isolated. Using the technique of Southern hybridization we have sublocalized the loci; ApoA1/C3, CD3D, ETS1, PBGD, THY1, D11S29, D11S34, and D11S147 to the region between the two breakpoints on chromosome 11 and V lambda I, V lambda VI, V lambda VII, and D22S10 to the region between the breakpoints on chromosome 22. Using anonymous DNA probes, we found that D22S9 and D22S24 map proximal to the constitutional breakpoint and that D22S15 and D22S32 map distal to the ES breakpoint on chromosome 22.     

Relationship of the human protooncogene CBL2 on 11q23 to the t(4;11), t(11;22), and t(11;14) breakpoints

A probe identifying CBL2, the human cellular homolog of the murine oncogene v-cbl and murine cellular protooncogene Cbl-2, and panels of rodent X human somatic cell hybrids were used to study the relationship of this protooncogene to translocations associated with acute leukemia, lymphoma, and Ewing sarcoma. CBL2 was mapped to 11q23 and found to translocate from chromosome 11 to 4 in an acute leukemia cell line possessing a t(4;11)(q21;q23) and from chromosome 11 to 14 in a B-cell lymphoma with a t(11;14)(q23;q32). In an Ewing sarcoma cell line with a t(11;22)(q23;q12), however, CBL2 remained on chromosome 11. Additional studies of other genes in the region of 11q23 allowed the following ordering of these genes and breakpoints: 11cen--q23--NCAM--CD3(E-D-G)--[t(11;14), t(4;11)]--(THY1, CBL2, ETS1)--t(11;22)--11qter. The gross structure of the CBL2 sequences examined was not altered by either of the flanking breakpoints. Given that the 5' and 3' ends of the CBL2 gene are not known and are probably not evaluated by the v-cbl probe, these results do not rule out the possibility of CBL2 involvement in the pathogenesis of a subset of acute leukemias possessing a t(4;11), B-cell lymphomas possessing a t(11;14), or Ewing sarcomas possessing a t(11;22).     

Mytilus edulis Histone Gene Clusters Containing Only H1 Genes

We isolated five different phage clones containing histone gene clusters with up to five H1 genes per phage clone from a Mytilus edulis genomic library. Among these H1 genes, nine gene types coding for five different H1 proteins have been identified. All H1 histone genes were located on repetitive restriction fragments with only slightly different sizes. The H1 coding regions show highly related sequences, suggesting that the multitude of H1 genes has evolved by gene duplication events. Core histone genes could not be found on these five Mytilus edulis genome fragments. Received: 28 July 1998 / Accepted: 17 May 1999     

神经粘附分子NCAM140 基因RNA干扰质粒的构建与鉴定

摘要目的：构建有效的小鼠神经粘附分子（NCAM140）基因的RNA 干扰（RNAi）质粒载体,为研究NCAM140参与的细胞信号通 路转导、其生物学作用以及以NCAM140 为靶点的基因治疗提供稳定转染的RNAi 质粒。方法：使用基因序列软件设计、筛选符 合公开文献筛选参数的4 条靶序列以及1条阴性对照序列,由上海吉玛技术有限公司合成。与载体质粒pGPU6/GFP/Neo 重组后, 分别命名为pSi-nca1、pSi-nca2、pSi-nca3、pSi-nca4和pSi-control。转染大肠杆菌感受态细胞。选择阳性克隆进行DNA 测序鉴定, Western blot方法进行干扰靶点的筛选。选取干扰效率最高的质粒转染MN9D 细胞,普通光学显微镜分别计数同一视野细胞总数 及GFP阳性细胞数,计算转染效率。结果：酶切和DNA 测序结果证实shRNA正确插入pGPU6/GFP/Neo 质粒;Western blot结果 显示与空质粒对照组比较,pSi-nca4 组细胞NCAM140 表达明显下调,细胞转染效率为62 %。结论：成功构建靶向小鼠 NCAM140 基因的RNAi 质粒,为NCAM140 参与的细胞信号通路的研究以及以NCAM140为靶点的基因治疗提供了稳定转染 细胞的干扰质粒,为研究其生物学作用奠定了分子生物学基础。     

Genomic analysis of the murine odorant receptor MOR28 cluster: a possible role of gene conversion in maintaining the olfactory map

Genomic analysis was performed for the murine odorant receptor (OR) genes. The MOR28 cluster on chromosome 14 was extensively studied. It contains six OR genes, MOR28, 10, 83, 29A, 29B and 30. The human homolog of this cluster is located on the human chromosome 14, and contains five OR genes, HOR28/10, 83, 29A, 29B and 30. Sequence comparison of these OR gene paralogs and orthologs suggests that the coding homologies are accounted for not only by recent gene duplication, but also by gene conversion among the coding sequences within the cluster. A possible role of gene conversion in the olfactory system is discussed in the context of the olfactory map.     

Sequence analysis of gene 11 equivalents from "short" and "super short" strains of rotavirus

The molecular basis for the aberrant migration pattern of the gene 11 equivalent in rotaviruses with "short" (human DS-1) and "super short" (human 69M and bovine VMRI) electropherotypes was investigated. The mRNAs of these viruses were synthesized in vitro, and the entire gene 11 equivalent of each of these viruses was sequenced with specific synthetic oligonucleotide primers. These sequences were compared with previously published sequences of "long" pattern rotavirus gene 11 segments. The increased lengths of the gene 11 equivalents of DS-1, 69M, and VMRI are due to a prolonged, 3' untranslated region in this gene segment. The 3' untranslated region of the VMRI gene 11 equivalent contains a clear duplication of a portion of its coding sequence. A stretch of 18 consecutive nucleotides within the 330-nucleotide, 3' untranslated region of 69M is identical to a section of UK coding sequence. The DS-1 and the remainder of the 69M 3'-end additional sequences are similar to each other, but neither is similar to any other currently available rotavirus gene sequence. This finding suggests that a process other than homologous duplication is involved in the evolution of these sequences. The widespread occurrence of human and animal rotaviruses with short and super short electropherotypes provides evidence that intragenic and possibly intergenic recombinational events associated with an error-prone viral RNA polymerase may play a role in increasing the genetic repertoire of rotaviruses.     

Characterization of rat brain NCAM mRNA using DNA oligonucleotide probes

A number of different isoforms of the neural cell adhesion molecule (NCAM) have been identified. The difference between these is due to alternative splicing of a single NCAM gene. In rat brain NCAM mRNAs with sizes of 7.4, 6.7, 5.2, 4.3 and 2.9 kb have been reported. We have synthesized six DNA oligonucleotides, that hybridize to different exons in the NCAM gene. Furthermore we have constructed three oligonucleotides, that exclusively hybridize to mRNAs lacking certain exons, by letting them consist of sequences adjacent to both sides of the splice sites. By means of these probes we have characterized the five NCAM mRNAs in rat brain.     

Complete nucleotide sequence of the haemagglutinin gene from a human influenza virus of the Hong Kong subtype.

The complete nucleotide sequence has been determined for a cloned double-stranded DNA copy of the haemagglutinin gene from the human influenza strain A/NT/60/68/29C, a laboratory-isolated variant of A/NT/60/68, an early strain of the Hong Kong subtype. The gene is 1765 nucleotides long and contains information sufficient to code for a protein of 566 amino acids, which includes a hydrophobic leader peptide (16 residues), HA1 (328), HA2 (221) and an arginine residue which joins the HA subunits. Comparison of the predicted amino acid sequence for 29C haemagglutinin with protein sequence data available for HA from other influenza strains shows that no potential coding information is lost by processing of the mRNA. A comparison of the amino acid sequences predicted from the gene sequences for 29C and fowl plague virus haemagglutinins, (1) indicates the extent to which changes can occur in the primary sequence of different regions of the protein, while maintaining essential structure and function.     

The complete genome sequence of the dominant Sinorhizobium meliloti field isolate SM11 extends the S. meliloti pan-genome

Isolates of the symbiotic nitrogen-fixing species Sinorhizobium meliloti usually contain a chromosome and two large megaplasmids encoding functions that are absolutely required for the specific interaction of the microsymbiont with corresponding host plants leading to an effective symbiosis. The complete genome sequence, including the megaplasmids pSmeSM11c (related to pSymA) and pSmeSM11d (related to pSymB), was established for the dominant, indigenous S. meliloti strain SM11 that had been isolated during a long-term field release experiment with genetically modified S. meliloti strains. The chromosome, the largest replicon of S. meliloti SM11, is 3,908,022 bp in size and codes for 3785 predicted protein coding sequences. The size of megaplasmid pSmeSM11c is 1,633,319 bp and it contains 1760 predicted protein coding sequences whereas megaplasmid pSmeSM11d is 1,632,395 bp in size and comprises 1548 predicted coding sequences. The gene content of the SM11 chromosome is quite similar to that of the reference strain S. meliloti Rm1021. Comparison of pSmeSM11c to pSymA of the reference strain revealed that many gene regions of these replicons are variable, supporting the assessment that pSymA is a major hot-spot for intra-specific differentiation. Plasmids pSymA and pSmeSM11c both encode unique genes. Large gene regions of pSmeSM11c are closely related to corresponding parts of Sinorhizobium medicae WSM419 plasmids. Moreover, pSmeSM11c encodes further novel gene regions, e.g. additional plasmid survival genes (partition, mobilisation and conjugative transfer genes), acdS encoding 1-aminocyclopropane-1-carboxylate deaminase involved in modulation of the phytohormone ethylene level and genes having predicted functions in degradative capabilities, stress response, amino acid metabolism and associated pathways. In contrast to Rm1021 pSymA and pSmeSM11c, megaplasmid pSymB of strain Rm1021 and pSmeSM11d are highly conserved showing extensive synteny with only few rearrangements. Most remarkably, pSmeSM11b contains a new gene cluster predicted to be involved in polysaccharide biosynthesis. Compilation of the S. meliloti SM11 genome sequence contributes to an extension of the S. meliloti pan-genome.