籼稻'93-11'一段基因组序列的完善及分析

超级杂交稻父本‘93-11＇的基因组序列测定的完成,为进行作物遗传改良和不同作物之间的比较基因组学研究提供了又一重要序列资源.但是,该基因组序列中还存在很多“缺口”,为使‘93-11＇的基因组序列更加精确,同时提供一些“缺口”填补策略和方法,本研究采用PCR扩增、回收克隆测序的方法对该基因组中一段长约160 kb、含有6个“缺口”的基因组序列进行了完善,并运用相关分子生物学和生物信息学软件进行了详细分析,结果表明：该6个“缺口”中,存在1个“缺口”估计错误,2个序列拼接错误;“缺口”主要位于非编码区,位于编码区的只有1个,其改变了对本处基因的注释,使此基因由原来的9个外显子增加为11个;填补“缺口”后,基因密度增加.     

籼稻‘93-11’一段基因组序列的完善及分析

超级杂交稻父本93-11的基因组序列测定的完成,为进行作物遗传改良和不同作物之间的比较基因组学研究提供了又一重要序列资源.但是,该基因组序列中还存在很多缺口",为使93-11的基因组序列更加精确,同时提供一些缺口"填补策略和方法,本研究采用PCR扩增、回收克隆测序的方法对该基因组中一段长约160kb、含有6个缺口"的基因组序列进行了完善,并运用相关分子生物学和生物信息学软件进行了详细分析,结果表明:该6个缺口"中,存在1个缺口"估计错误,2个序列拼接错误;缺口"主要位于非编码区,位于编码区的只有1个,其改变了对本处基因的注释,使此基因由原来的9个外显子增加为11个;填补缺口"后,基因密度增加.     

Structure of the murine lactotransferrin gene is similar to the structure of other transferrin-encoding genes and shares a putative regulatory region with the murine myeloperoxidase gene

The structure and nucleotide sequence of the murine lactotransferrin-encoding gene (LTF) deduced partly by direct sequencing of genomic clones in the λ phage vector and partly by enzymatic amplification of genomic DNA segments primed with the oligodeoxyribonucleic primers homologous to the cDNA sequence. The λ phage clones contained the 5′ half of the gene corresponding to the first eight exons and an incomplete ninth exon interrupted by eight introns. Genomic clones corresponding to the 3′ half of the LTF gene could not be obtained on repeated attempts from two different mouse genomic libraries, suggesting the possible presence of unclonable sequences in this part of the gene. Hence, PCR was used to clone the rest of the gene. Four out of the presumed eight remaining introns were cloned along with the flanking exons using PCR. Comparison of the structure of the LTF gene with those of the two other known transferrin-encoding genes, human serum transferrin-encoding gene and chicken ovotransferrin-encoding gene reveals that all three genes have a very similar intron-exon distribution pattern. The hypothesis that the present-day transferrin-encoding genes have originated from duplication of a common ancestral gene is confirmed here at the gene level. An interesting finding is the identification of a region of shared nucleotides between the 5′ flanking regions of the murine LTF and myeloperoxidase-encoding genes, the two genes expressed specifically in neutrophilic granulocytes.     

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.     

Isolation of the osteonectin gene: evidence that a variable region of the osteonectin molecule is encoded within one exon

A complementary DNA clone for bovine osteonectin was used to isolate the osteonectin gene from two libraries of bovine genomic DNA fragments. Two overlapping clones were obtained whose relationship was determined by restriction mapping and sequence analysis. The two clones contain the entire osteonectin coding region spanning approximately 11 kilobases of genomic DNA. The coding region of the gene was determined, by electron microscopy and DNA sequencing, to reside in nine exons. In addition, there is at least one 5' exon interrupted by an intron in the 5'-nontranslated sequence of the gene. Excluding this 5' exon and the 3'-terminal exon, the exons are small and approximately uniform in size, averaging 130 +/- 17 base pairs. Three of the exons at the 5' end of the gene were sequenced and appear to encode discrete protein domains. For example, the putative exon 2 contains the coding region for the leader peptide of the molecule. The amino-terminal protein sequence was determined for osteonectin extracted from human, rabbit, and chicken bone and compared with those for bovine, mouse, and pig osteonectin. These data suggest that osteonectin is highly conserved between species, interspecies changes being seen primarily at the amino terminus of the protein and specifically in the region encoded by putative exon 3 in the bovine gene.     

Characterization and SNP Identification of Part of the Goat Melanophilin Gene

The melanophilin (MLPH) gene has been characterized as the candidate gene for dilute coat color in some species, but little is known about it in the goat. In this study, part of the genomic DNA sequence (19,289 bp) containing the whole coding region of the MLPH gene from goat, as well as from sheep, was determined. We found 16 exons and 15 introns; the coding region was 1767 bp distributed in 15 exons (2–16). In sheep, the length of part of the genomic DNA sequence was 16,988 bp, with 16 exons and 15 introns, and the coding region was 1833 bp, distributed in 15 exons (2–16). Dozens of SNPs as well as some noticeable motifs in the goat MLPH gene were found during the process of sequencing and polymorphism screening. Based on the SSR Tool, three simple sequence repeat motifs were detected in the goat and sheep DNA sequences. Compared with cattle, we found insertions of 4 amino acids in goats and 26 amino acids in sheep.     

用基因组DNA剪接技术克隆SIgA相关基因

目的：克隆分泌型IgA（SIgA）相关基因--J链基因(IgJ)、多聚免疫球蛋白受体基因（pIgR）和IgA重链恒定区基因（IGHA）,为进一步构建SIgA真核表达质粒奠定基础。方法：采用本室建立的"基因组DNA剪接"技术,根据已发表的IgJ、pIgR和IGHA的核苷酸序列,通过计算机软件分别设计各个基因片段外显子的优化引物,从人外周血基因组DNA中直接扩增各基因的外显子序列;然后人工设计融合相邻外显子的融合引物,采用重叠PCR技术,把各基因片段的外显子串联起来形成全长编码序列,完成基因组DNA的体外剪接。扩增的PCR产物纯化后克隆到pGEM-T Easy Vector中,通过DNA测序对阳性克隆进行分析鉴定。结果：PCR扩增的IgJ、pIgR和IGHA基因与预期大小一致;测序结果表明本实验获得的上述基因与GenBank中的目标基因序列完全一致。结论：本文通过基因组DNA剪接技术成功克隆人类SIgA三个相关基因,提示此技术是合成多外显子cDNA的有效手段。     

Cloning and characterization of the carp prolactin gene

A carp genomic DNA clone containing the carp prolactin (Prl) gene was isolated with carp Prl cDNA as a probe. The organization of the carp Prl gene was determined by restriction nuclease mapping and nucleotide sequencing. The Prl gene comprises approx. 2.8 kilobasepairs (kb) of DNA including the 5'-flanking region, five exons, four introns and the 3'-flanking region. Analysis of the 5'-flanking region reveals (1) the sequence TATATAAT at positions -38 to -31 upstream from the cap site which was found to be a guanine residue, and (2) the palindrome, CTCATTGCATATACAAATGAG at positions -79 to -59. The carp Prl gene matches with the reported cDNA except for one difference in coding region and five in the 3'-flanking region, while the encoded amino acid sequences are identical. The arrangement of exons and introns is very similar to that seen in carp GH as well as mammalian Prl, which, however, have much longer introns.     

Discovery of Candidate Disease Genes in ENU–Induced Mouse Mutants by Large-Scale Sequencing,Including a Splice-Site Mutation in Nucleoredoxin

An accurate and precisely annotated genome assembly is a fundamental requirement for functional genomic analysis. Here, the complete DNA sequence and gene annotation of mouse Chromosome 11 was used to test the efficacy of large-scale sequencing for mutation identification. We re-sequenced the 14,000 annotated exons and boundaries from over 900 genes in 41 recessive mutant mouse lines that were isolated in an N-ethyl-N-nitrosourea (ENU) mutation screen targeted to mouse Chromosome 11. Fifty-nine sequence variants were identified in 55 genes from 31 mutant lines. 39% of the lesions lie in coding sequences and create primarily missense mutations. The other 61% lie in noncoding regions, many of them in highly conserved sequences. A lesion in the perinatal lethal line l11Jus13 alters a consensus splice site of nucleoredoxin (Nxn), inserting 10 amino acids into the resulting protein. We conclude that point mutations can be accurately and sensitively recovered by large-scale sequencing, and that conserved noncoding regions should be included for disease mutation identification. Only seven of the candidate genes we report have been previously targeted by mutation in mice or rats, showing that despite ongoing efforts to functionally annotate genes in the mammalian genome, an enormous gap remains between phenotype and function. Our data show that the classical positional mapping approach of disease mutation identification can be extended to large target regions using high-throughput sequencing.     

Structural organization of the gene for the alpha 1 chain of human type IV collagen

The complete exon size and distribution pattern in the gene for the alpha 1 chain of human type IV collagen was determined. Clones covering 145 kilobases (kb) of genomic DNA including 100 kb of the gene itself as well as 25 kb upstream and 20 kb downstream of the gene sequences, respectively, were isolated from lambda phage and cosmid libraries. The overall gene structure was determined by endonuclease restriction mapping and R-loop analyses and all exon sizes by nucleotide sequencing. The characterized clones contained all the coding sequences except for exon 2 whose sequence was determined after its amplification by the polymerase chain reaction. There were four gaps in the intron sequences; the exact size of the gene is unknown. The entire gene is at least 100 kb in size and contains 52 exons whose size distribution is completely different from that of the genes for fibrillar collagens. In the -Gly-X-Y- coding region there are three exons of 99, 90, and 45 base pairs (bp) each and two exons of 27, 36, 42, 51, 54, 63, and 84 bp each. The rest of the exons have sizes between 71 and 192 bp in the collagenous region. About one-half of the -Gly-X-Y- repeat coding exons start with the second base for the codon of glycine, whereas the other half starts (with two exceptions) with a complete glycine codon. The distribution of split versus unsplit codons is uneven in that the first 19 exons of the gene start with a complete codon. The gene contains repetitive sequences in several regions. A 185-nucleotide segment containing 40 copies of CCT flanked by poly(C) and poly(T) sequences was shown to be located adjacent to an exon. The gene has previously been shown to be located head-to-head to the alpha 2(IV) collagen gene at the distal end of the long arm of chromosome 13, such that the first exons of the two genes are separated by as little as 42 bp (P?schl, E., Pollner, R., and Kühn, K. (1988) EMBOJ. 7,2687-2695; Soininen, R., Huotari, M., Hostikka, S. L., Prockop, D. J., and Tryggvason, K. (1988) J. Biol. Chem. 263, 17217-17220). The results demonstrate that the human alpha 1(IV) collagen gene has a structure distinctly different from the genes for fibrillar collagens and also that it is considerably larger than any collagen gene characterized to date.     

Rapid and efficient FBN1 mutation detection using automated sample preparation and direct sequencing as the primary strategy

Mutations in the fibrillin-1 (FBN1) gene cause Marfan syndrome (MFS) and the other type-1 fibrillinopathies. Finding these mutations is a major challenge considering that the FBN1 gene has a coding region of 8,600 base pairs divided into 65 exons. Most of the more than 600 known mutations have been identified using a mutation scanning method prior to sequencing of fragments with a suspected mutation. However, it is not obvious that these screening methods are ideal, considering cost, efficiency, and sensitivity. We have sequenced the entire FBN1 coding sequence and flanking intronic sequences in samples from 105 patients with suspected MFS, taking advantage of robotic devices, which reduce the cost of supplies and the quantity of manual work. In addition, automation avoids many tedious steps, thus reducing the opportunity for human error. Automated assembling of PCR, purification of PCR products, and assembly of sequencing reactions resulted in completion of the FBN1 sequence in half of the time needed for the manual protocol. Mutations were identified in 69 individuals. The mutation detection rate (76%), types, and genetic distribution of mutations resemble the findings in other MFS populations. We conclude that automated sequencing using the robotic systems is well suited as a primary strategy for diagnostic mutation identification in FBN1.     

Optimized Heterologous Expression of the Polymorphic Human Glutathione Transferase M1-1 Based on Silent Mutations in the Corresponding cDNA

An expression clone for large-scale production of the polymorphic human glutathione transferase (GST) M1-1 has been developed. Heterologous expression inEscherichia coliafforded a yield of 100 mg of GST M1-1 per 3 liters of culture medium, corresponding to an approximately 10-fold increased yield compared to the parental expression construct. Overproduction of the enzyme was dependent on the codon usage in the 5′ region of the DNA sequence encoding glutathione transferase M1-1. High-level expression clones were generated by a combination of random silent mutations in selected wobble positions in the coding sequence and immunoselection of clones from the library of random mutants. The strategy used is generally applicable for the production of recombinant proteins provided that a suitable selection procedure is available for identifying the desired mutants.     

Chicken vigilin gene organization and expression pattern. The domain structure of the protein is reflected by the exon structure.

Chicken vigilin was identified as a member of an evolutionary-conserved protein family with a unique repetitive domain structure. 14 tandemly repeated domains are found in chicken vigilin, all of which consist of a conserved sequence motif (subdomain A) and a potential alpha-helical region (subdomain B) [1]. We have established the physical structure of the chicken vigilin gene by restriction-fragment analysis and DNA sequencing of overlapping clones isolated from a phage lambda genomic DNA library. The chicken vigilin gene is a single-copy gene with a total of 27 exons which are distributed over a region of some 22 kbp. Exon 1 codes for a portion of the 5' untranslated region, exon 2 contains the translation start point and forms, along with exons 3 and 4, the N-terminal non-domain region. Exons 5-25 encode the vigilin domains 1-14 and the remaining exons 26 and 27 contain the non-domain C-terminal as well as the untranslated regions. The domain structure of the protein is reflected in the positioning of introns which demarcate individual domains. While domains 1-3 and 8-10 are each encoded by a single exon (5-7, 16-18); all other domains are contained in a set of two exons which are separated by introns interspersed at variable positions of the DNA segment coding for the conserved sequence motif. In conclusion, the data presented suggest that the chicken vigilin gene evolved by amplification of a primordial exon unit coding for the fundamental bipartite vigilin domain.     

The Bos taurus casein genes. Isolation and characterization of the kappa-casein gene

A region spanning 25 kb of genomic DNA containing the kappa-casein gene, has been isolated from two genomic libraries in EMBL3 and EMBL4 phage vectors. Five phage clones containing kappa-casein gene have been found. Gene organisation has been determined using restriction mapping and a partial sequencing the 5' and 3' flanking regions. The kappa-casein gene includes 5 exons, the first of them coding for 64 nucleotides from the 5' untranslated mRNA zone. The gene is 12.5 kb long, which is almost 16 times longer than the corresponding mRNA. The first intron spans 2.5 kb, the second is the largest one and spans 5.5 kb. The 5' flanking region sequence has been analysed; it contains a TATA box from -30 to -25 bp, somewhat different from the canonic sequence, and a CAAT box at -80 bp.     

Amplification and direct nucleotide sequencing of cDNA from the lysate of low numbers of diploid human cells

The polymerase chain reaction technique is widely employed to amplify short segments of genomic DNA to determine if a specific change has occurred. However, some investigators need to sequence the entire coding region of mammalian genes, e.g., cellular ras genes or the gene encoding hypoxanthine (guanine) phosphoribosyl transferase (HPRT), to determine what specific changes have occurred. To do so, they isolate RNA from large populations of cells, amplify cDNA from the gene of interest, subclone the product, and sequence two or more isolates to determine the common mutation. We have developed a method to simplify this procedure by copying mRNA of the hprt gene directly from the lysate of a clone of mutant diploid human fibroblasts (e.g., 100 cells). We amplified the first and second strand of the cDNA of the gene of interest 10(10)- to 10(11)-fold, obtained 5 to 10 micrograms of DNA in less than 10 h, and sequenced the coding region directly without the need for RNA extraction or DNA template purification. By our method cDNA can be amplified directly from the lysate of just one human cell, but to avoid detecting random changes introduced by the polymerase, we lysed approx. 200 cells from a clone, each containing the identical mutation, amplified the cDNA, and determined the consensus sequence by direct nucleotide sequencing.     

The M1- and M2-type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing

The complete nucleotide sequences of rat M1- and M2-type pyruvate kinase mRNAs were determined by sequencing the cDNAs and by analyses of S1 nuclease mapping and primer extension. The sequences have an identical molecular size of about 2220 nucleotides excluding a poly(A) tail and include 1593-nucleotide coding region. Their nucleotide sequences are identical except for 160-nucleotide sequences within the coding regions. The amino acid sequences of the M1- and M2-type subunits deduced from the cDNA sequences differ by only 45 residues within domain C, which constitutes the main region responsible for intersubunit contact. The sequence of this region of the M2-type shows higher homology than that of the M1-type with the corresponding sequence of the L-type. Since the M2- and L-types are allosteric enzymes, unlike to the M1-type, the residues common to the M2- and L-types, but not the M1-type may be important for mediating the allosteric properties. Genomic clones encoding both M1- and M2-type isozyme mRNAs were isolated. By partial sequence analysis of a clone lambda MPK37 four exons were identified, of which two adjacent exons coded the M1- and M2-specific sequences, respectively. The two remaining exons present downstream coded amino acids common to the two isozymes. Thus, we conclude that the M1- and M2-type isozymes of pyruvate kinase are produced from the same gene probably by alternative RNA splicing.     

Random oligonucleotide mutagenesis: application to a large protein coding sequence of a major histocompatibility complex class I gene, H-2DP.

We have used random oligonucleotide mutagenesis (or saturation mutagenesis) to create a library of point mutations in the alpha 1 protein domain of a Major Histocompatibility Complex (MHC) molecule. This protein domain is critical for T cell and B cell recognition. We altered the MHC class I H-2DP gene sequence such that synthetic mutant alpha 1 exons (270 bp of coding sequence), which contain mutations identified by sequence analysis, can replace the wild type alpha 1 exon. The synthetic exons were constructed from twelve overlapping oligonucleotides which contained an average of 1.3 random point mutations per intact exon. DNA sequence analysis of mutant alpha 1 exons has shown a point mutant distribution that fits a Poisson distribution, and thus emphasizes the utility of this mutagenesis technique to "scan" a large protein sequence for important mutations. We report our use of saturation mutagenesis to scan an entire exon of the H-2DP gene, a cassette strategy to replace the wild type alpha 1 exon with individual mutant alpha 1 exons, and analysis of mutant molecules expressed on the surface of transfected mouse L cells.     

Cloning and characterization of a third type of human alpha-amylase gene, AMY2B

We have previously reported concerning the existence of a third type of human alpha-amylase gene, AMY3 [Emi et al., Gene 62 (1988) 229-235; Tomita et al., Gene 76 (1989) 11-18], which is expressed in a lung carcinoid tissue, and differs in nucleotide sequence from the two previously characterized human alpha-amylase genes coding for salivary and pancreatic isozymes, termed AMY1 and AMY2, respectively. Here, we rename this gene AMY2B to coincide with the designation by Gumucio et al. [Mol. Cell Biol. 8 (1988) 1197-1205] and describe its genetic properties as revealed by sequencing studies. It consists of ten major exons whose sequences are highly homologous to those of AMY1 and AMY2. Not only the exons, but also most of the introns seem to be highly conserved, as judged from physical mapping data. The AMY2B gene identified from mRNA in a lung carcinoid tissue has at least two additional untranslated exons in its 5' region; hence the promoter lies far upstream relative to the other two AMY genes.