水稻杂种与亲本间差异表达cDNA片段S 600的分离克隆

采用cDNA-AFLP技术分离克隆了水稻杂种与亲本间差异表达基因片段．S600,Northern杂交结果表明：在分蘖期和始穗期,S600在杂种和父本中表达丰度均较高,而在母本中表达丰度相对较低。S600在分蘖期和始穗期表达量不同,暗示了该基因的表达还受到发育时期的调节。同源搜索结果表明S600片段是水稻SBPase的部分编码序列。为了获得完整编码序列,以S600序列检索梗稻日本晴cDNA数据库,获得了两个高度同源(99％)且功能未知的全长cDNA克隆(AK062089和AK065773)。序列分析表明它们均包含一个相同的1179bp的开放阅读框,编码392个氨基酸组成的水稻SBPase前体,其中包含有与底物结合、氧化还原调节有关的保守氨基酸残基。检索发现该基因在水稻日本晴基因组中只有单个座位。     

水稻杂种与亲本间差异表达cDNA片段S600的分离克隆

采用cDNA-AFLP技术分离克隆了水稻杂种与亲本间差异表达基因片段S600。Northern杂交结果表明:在分蘖期和始穗期,S600在杂种和父本中表达丰度均较高,而在母本中表达丰度相对较低。S600在分蘖期和始穗期表达量不同,暗示了该基因的表达还受到发育时期的调节。同源搜索结果表明S600片段是水稻SBPase的部分编码序列。为了获得完整编码序列,以S600序列检索粳稻日本晴cDNA数据库,获得了两个高度同源(99%)且功能未知的全长cDNA克隆(AK062089和AK065773)。序列分析表明它们均包含一个相同的1179bp的开放阅读框,编码392个氨基酸组成的水稻SBPase前体,其中包含有与底物结合、氧化还原调节有关的保守氨基酸残基。检索发现该基因在水稻日本晴基因组中只有单个座位。     

Evolution of the fibronectin gene. Exon structure of cell attachment domain

Genomic DNA coding for human fibronectin was identified from a human genomic library by screening with a cDNA clone that specifies the cell attachment domain in human fibronectin. Two clones which together provided more than 22 kilobase pairs of the fibronectin gene were isolated. The exons in this region correspond to approximately 40% of the coding region in the fibronectin gene. They code for the middle region of the polypeptide which consists of homologous repeating segments of about 90 amino acids called type III homologies. Nucleotide sequence of the portion of the gene corresponding to the cell attachment domain showed that the Arg-Gly-Asp-Ser cell attachment site is encoded within a 165-base pair exon. This exon, together with a 117-base pair exon codes for a homology unit. Analysis of the exon/intron organization in some of the neighboring homology units indicated a similar 2-exon structure. An exception to this pattern is that a single large exon codes for a type III homology unit that, due to alternative mRNA splicing, exists in some but not all fibronectin polypeptides. The introns separating the coding sequences for the type III homology units are located in conserved positions whereas the introns that interrupt the coding sequence within the units are in a variable position generating variations in the size of the homologous exons. This exon/intron organization suggests that the type III homology region of the fibronectin gene has evolved by a series of gene duplications of a primordial gene consisting of two exons. Specification of one of these homology units to the cell attachment domain has occurred within this exon/intron arrangement.     

Structure of the human genomic region homologous to the bovine prochymosin-encoding gene

Two human genomic libraries were probed with bovine prochymosin (bPC) cDNA. Recombinant clones covering a genomic region homologous to the entire coding region and flanking sequences of the bPC gene were isolated. Human sequences homologous to exons of the bPC gene are distributed in a DNA fragment of 10 kb. Alignment of the human sequences and the exons of bPC reveals that the human 'exons' 1-3, 5 and 7-9 have sizes identical to the corresponding bovine exons, but a nucleotide (nt) has been deleted in the human exon 4 and two nt in the human exon 6. The aligned human sequence and the coding part of bPC gene share 82% nt homology, the value ranging, in separate exons, from 76 (exon 1) to 84% (exons 5 and 6). 150 bp of 5'-flanking sequence of the human gene has 75% homology to the corresponding region of bPC gene and contains a TATA-box in a similar position. A 1-nt deletion in the human exon 4 would shift the translational reading frame of a putative human PC mRNA relative to bPC mRNA, and result in an in-phase terminator spanning codons 163 and 164 in bPC mRNA. Another terminator in-phase with the amino-acid sequence encoded by the bPC gene occurs in the human exon 5 and the second frameshift mutation in exon 6. Thus, the nt sequence analysis of the human genomic region has revealed the presence of mutations that have rendered it unable to produce a full-length protein homologous to bPC and, therefore, we refer to this gene as a human prochymosin pseudogene (hPC psi). Blot-hybridization analysis of human genomic DNA indicates that hPC psi is a single gene in the human genome.     

A guinea-pig hereditary cataract contains a splice-site deletion in a crystallin gene.

A congenital cataract present in guinea pigs provided a unique opportunity to study a hereditary lens disease at the molecular level. zeta-Crystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several zeta-crystallin cDNA clones were isolated from a cataractous lens library and found to contain a 102-bp deletion towards the 3' end of the coding region. This deletion does not interfere with the reading frame but results in a protein 34 amino acids shorter. Sequence analysis of a normal genomic zeta-crystallin clone revealed that the missing 102-bp fragment corresponds to an entire exon (exon 7). PCR analysis of the genomic DNA isolated from cataractous animals showed that exon 7, though missing from the mRNA, is intact in the cataractous genome. Further sequence analysis of the zeta-crystallin gene disclosed a dinucleotide deletion of the universal AG at the acceptor splice-site of intron 6 of the mutant gene. The presence of this mutation results in the skipping of exon 7 during the mRNA processing which in turn results in the altered zeta-crystallin protein. This is the first time a genomic mutation in an enzyme/crystallin gene has been directly linked to a congenital cataract.