The Mitochondrial Genome Organization of a Maize Fertile Cmst Revertant Line Is Generated through Recombination between Two Sets of Repeats

The mitochondrial genome (mtDNA) organization from a fertile revertant line (V3) derived from the maize cytoplasmic male sterile type T (cmsT) callus tissue culture has been determined. We report that the sequence complexity can be mapped on to a circular ``master chromosome' of 705 kb which includes a duplication of 165 kb of DNA when compared to its male sterile progenitor. Associated with this event is also a 0.423-kb deletion, which removed the cmsT-associated urf13 gene. As found for the maize normal type (N) and cmsT mitochondrial genomes, the V3 master chromosome also exists as a multipartite structure generated by recombination through repeated sequences.     

黑曲霉糖化酶高产和低产菌株糖化酶基因调控区的克隆及其分析比较

以PCR合成的糖化酶高产菌株黑曲霉(Asp. Niger)T21糖化酶基因5’近端非编码区588bp(EcoRI-BamHI)的序列为探针,从T21染色体DNA中克隆到近2.0kb的糖化酶基因5’端非编码区序列,并以此序列为探针从糖化酶低产菌株黑曲霉3.795(T21的诱变出发株)的染色体DNA中克隆到1.5kb的糖化酶基因5’端非编码区序列。该二序列的分析测定结果表明,其结构特征与文献报道的黑曲霉糖化酶基因5’端非编码区的基本一致,被称为“核心启动子”(Core promoter)的TATAAAT框及GCAAT框,分别在翻译起始点的-109bp及-178bp处。此外,在曲霉amdS,amyB基因中已发现有调控功能的CCAAT序列存在于-449bp和-799bp处。高产和低产菌株糖化酶基因5’端非编码区序列的分析比较结果表明,有9个部位的碱基发生了变化。此实验结果为进一步研究黑曲霉糖化酶基因在转录水平上的调控规律打下了基础。     

A rearranged DNA sequence possibly related to the translocation of immunoglobulin gene segments.

A 5.3 kb EcoRI fragment (T3, abbreviations in ref. 2) has been cloned from DNA of a kappa light chain producing mouse myeloma. The fragment hybridizes to the k' flanking sequences of the J1 gene segment but not to C gene sequences of kappa light chain DNA. Restriction nuclease mapping and partial nucleotide sequencing showed that the fragment consists of sequences from the 5' side of the J1 and form the 3' side of a V gene segment, which apparently had been linked in a genomic rearrangement process. These rearranged flanking sequences are not the flanking sequences of the V and J gene segments which had been joined to form the two kappa light chain genes of the myeloma. Fragments with the hybridization properties of T3 have been found also in two other kappa and one lambda chain producing myelomas. The linking of flanking sequences in the myeloma genome is discussed with respect to the mechanism of recombination between V and J gene segments.     

The human transferrin gene: 5' region contains conserved sequences which match the control elements regulated by heavy metals, glucocorticoids and acute phase reaction

Transferrin is a major plasma protein that transports iron to proliferating cells throughout the body. A clone containing the 5' region of the human transferrin gene has been isolated and characterized. A 14 kb EcoRI fragment was identified that contained the first 8 exons of the transferrin gene and 3.6 kb of its 5' flanking region. Conserved sequences identical or homologous to regulatory elements responding to heavy metals, glucocorticoid receptor and a putative acute phase reaction signal were identified in the 5'flanking region and intron 1. Also, the regulatory region of the transferrin gene contains a 14-bp sequence which closely matches sequences found in the interleukin-2 and gamma-interferon genes. All three genes are expressed by T lymphocytes before proliferation. A secondary loop structure similar to that proposed for the ovotransferrin gene can be formed by sequences in the 5' untranslated region of the transferrin mRNA.     

A fertile revertant from petaloid cytoplasmic male-sterile carrot has a rearranged mitochondrial genome

 A spontaneously derived fertile plant was recovered from a petaloid cytoplasmic male-sterile (CMS) carrot inbred line. Genetic analysis indicated a single nuclear gene was responsible for the restoration to fertility. Within a family segregating for the nuclear restorer in combination with the sterility-inducing cytoplasm, fertile plants were recovered that could not restore fertility when crossed to sterile genotypes. Genetic analysis indicated cytoplasmic reversion for fertility, and Southern analysis, comparing mtDNA organization of the fertile revertant and its CMS progenitor, identified mitochondrial genome rearrangements. Hybridization of cosmids representing a 108-kb subgenomic circle of the sterile line to DNA of a fertile maintainer and fertile revertant lines indicated a similar mtDNA organization for these genotypes that was distinct from that of the sterile line. Six restriction fragments totalling 43.2 kb were common to the fertile maintainer and revertant and absent in the sterile; other restriction fragments totalling 38.2 kb were present only for the sterile line. Unique fragments of low stoichiometry, two for the fertile maintainer and three for the revertant, distinguished these lines. The reversion to fertility in the sterile line could have resulted from the amplification of a mitochondrial submolar genome highly homologous to that found in the fertile maintainer line. Received: 4 October 1997/Accepted: 12 December 1997     

青花菜雄性不育相关基因BoDHAR的克隆与表达分析

以一个与甘蓝显性核不育相关的差异表达片段的序列为信息探针,通过在NCBI与TAIR网站数据库中进行同源EST序列搜索,经人工拼接、RT-PCR、PCR克隆与序列分析,获得了青花菜脱氢抗坏血酸还原酶DHARdehydroascorbatereductase基因的cDNA与DNA全长序列,命名为BoDHAR。并利用双链接头介导PCR的染色体步行技术(genomewalking)克隆了其上游644bp的5′端序列。所获的BoDHAR基因全长1486bp,存在两个内含子,DNA编码区序列633bp,编码210个氨基酸;序列分析表明BoDHAR与同源基因AT1G19570.1cDNA序列有82.3%的一致性,推导的氨基酸序列有79.6%的一致性;编码的水溶性蛋白存在多个磷酸化位点;5′端上游区存在明显的转录调控序列。半定量RT-PCR结果表明BoDHAR在可育系花蕾中的表达量明显高于不育系花蕾,在花药中的表达明显高于其它部位。     

Structural differences in the renin gene of Dahl salt-sensitive and salt-resistant rats

Genomic libraries in lambda EMBL4 phage were constructed from both inbred Dahl salt-hypertension-sensitive (S) and inbred Dahl salt-hypertension-resistant (R) rats. Overlapping clones containing the renin genes were isolated from these libraries by screening with a renin cDNA probe. Clones were characterized by a combination of restriction mapping and Southern blot analysis. The results showed that the S-rat renin gene is remarkably different from the R-rat renin gene. The major differences are 1) a 1.2-kilobase (kb) insertion in the first intron of the S-gene which accounts for most of the restriction fragment length polymorphisms found in the renin genes between S and R strains, such as those generated with Bg/II [2.7 kb (S)/1.5 kb (R)], EcoRI [6.4 kb (S)/5.2 kb (R)], and HindIII [9.6 kb (S)/8.4 kb (R)]; 2) an additional HindIII site located at the 3' end of the R-gene which accounts for another HindIII restriction fragment length polymorphisms [25 kb (S)/22 kb, 3.4 kb (R)]; 3) two SmaI sites at the 5' flanking region of the first exon of the S-gene, whereas there is only one SmaI site in the corresponding region of the R-gene; and 4) three AvaI sites in the first intron of the S-gene in contrast to two AvaI sites in the same region of the R-gene These differences in the renin genes of Dahl rats might affect renin gene expression, which could account for the known strain differences in plasma and tissue renin activities. These structural studies provide a basis for genetic investigation into the relationship of the renin gene to blood pressure in Dahl rats.     

The 5'' flanking region of human epsilon-globin gene.

The structural analysis of the 2.0 kb region upstream from the epsilon-globin gene has been carried out. A genomic DNA map around the gene was worked out in some detail to ensure that the cloned DNA was representative of the actual chromosomal arrangement. Furthermore, a new technique was developed to precisely map a reiterated DNA sequence present 1.5 kb to the 5' side of the gene. The complete nucleotide sequence of the 2.0 kb 5' flanking region was then determined and overlapped with the gene. The sequence included the reiterated DNA sequence which is homologous to the so-called AluI family of repeats. Unusual stretches of sequence 50 nucleotides long, where A + T represent about 90% of the bases, are present at both the 5' and 3' sides of the repeat.     

粘虫核型多角体病毒一个新基因的序列和结构研究

本文报道ＴｓＮＰＶＤＮＡＥｃｏＲＶ／ＸｈｏＩ的５．５ｋｂ片段的克隆及其物理图谱。测定了其中一个８１９ｂＰ片段的全序列。在长３４６ｂｐ的完整ＯＲＦ的５’端除有ＴＡＴＡｂｏｘ和ＣＡＡＴｂｏｘ以外,还发现有典型的早期基因启动子元件ＡＣＧＴ和ＧＣ单元以及晚期基因的转录起始保守序列ＴＴＡＡＧ。预测的ＯＲＦ产物为１１４个氨基酸、分子量为１３ｋＤ的蛋白质,故命名为ｐ１３蛋白。在ｐ１３基因的Ｃ端和Ｎ端分别有一个亮氨酸拉链和两个类亮氨酸拉链结构（我们称为亮氨酸转型结构和ＬＶＴ重复结构〕．从终止密码起有一个双发卡环结构,ｐ１３基因的５’端调控单元及其排列与ＢｍＮＰＶ和ＡｃＮＰＶ的细胞序死抑制基因（ｐ３５）十分相似,但ｐ１３基因与已经发现的杆状病毒基因序列和氨基酸序列没有同源性．因此,这是一个新的早晚期基因,其调控元件可能具有重要功能。     

Mitochondrial DNA rearrangements in Pennisetum associated with reversion from cytoplasmic male sterility to fertility

Endonuclease restriction fragment patterns of Pennisetum americanum L. mitochondrial DNAs (mtDNAs) from a cytoplasmic male-sterile (CMS-A1), fertile revertants and a normal fertile cytoplasm were variable, while chloroplast DNA from those lines lacked variation. Comparisons between mtDNAs of CMS-A1 (parental) and fertile revertant lines revealed the presence of a unique 4.7 kbp PstI fragment in the sterile line that was not detected in any of the revertant lines. A 9.7 kbp PstI fragment was found in all of the revertants, but not in the CMS-A1. Neither of those fragments was found in the normal cytoplasm mtDNA. Hybridization studies revealed two sets of multiple homologies: 1) the 4.7 kbp fragment had homology with a 10.9 kbp and a 13.6 kbp fragment; and 2) the 9.7 kbp fragment was homologous with the 13.6 kbp fragment. The presence of those two repeated mitochondrial sequences on the altered fragments suggests that they may be involved in the recombinational associated events with reversion from CMS to fertility in P. americanum.Florida Agricultural Experiment Station Journal Series No.7797.     

Cloning and expression of the rfe–rff gene cluster of Escherichia coli

We have cloned a 13 kb Escherichia coli DNA fragment which complemented the rfe mutation to recover the biosynthesis of E. coli O9 polysaccharide. Using Tn5 insertion inactivation, the rfe gene was localized at the 1.5 kb HindIII-EcoRI region flanking the rho gene. We constructed an rfe-deficient E. coli K-12 mutant by site-directed inactivation using a DNA fragment of the cloned 1.5 kb rfe gene. This also confirmed the presence of the rfe gene in the 1.5 kb region. By simultaneous introduction of both the rfe plasmid and the plasmid of our previously cloned E. coli O9 rfb into this rfe mutant, we succeeded in achieving in vivo reconstitution of O9 polysaccharide biosynthesis. From sequence analysis of the rfe gene, a putative promoter followed by an open reading frame (ORF) was identified downstream of the rho gene. This ORF coincided with the position of the rfe gene determined by Tn5 analysis and site-directed mutagenesis. Furthermore, we identified the rff genes in the 10.5 kb DNA flanking the rfe gene. We recognized at least two functional domains on this cloned rff region. Region I complemented a newly found K-12 rff mutant, A238, to synthesize the enterobacterial common antigen (ECA). Deletion of region II resulted in the synthesis of ECAs with shorter sugar chains. When the 10.5 kb rff genes of the plasmid were inactivated by either deletion or Tn5 insertion, the plasmid lost its ability to give rise to transformants of the rfe mutants.     

Recovery of Heritable, Transposon-Induced, Mutant Alleles of the Rf2 Nuclear Restorer of T-Cytoplasm Maize

T (Texas) cytoplasm is associated with a mitochondrial disruption that is phenotypically expressed during microsporogenesis resulting in male sterility. Restoration of pollen fertility in T-cytoplasm maize is controlled by dominant alleles at two unlinked, complementary, nuclear-encoded genes, rf1 and rf2. As a first step in the molecular isolation of the rf2 gene, 178,300 gametes derived from plants that carried the Mutator, Cy or Spm transposon families were screened for rf2 mutant alleles (rf2-m) via their inability to restore pollen fertility to T-cytoplasm male-sterile maize. Seven heritable rf2-m alleles were recovered from these transposon populations. Pedigrees and restriction fragment length polymorphism (RFLP)-based analyses indicated that all seven rf2-m alleles were derived independently. The ability to obtain rf2-m derivatives from Rf2 suggests that Rf2 alleles produce a functional product necessary to restore pollen fertility to cmsT. Molecular markers flanking the rf1 and rf2 loci were used to decipher segregation patterns in progenies segregating for the rf2-m alleles. These analyses provided preliminary evidence of a weak, third restorer gene of cmsT that can substitute for Rf1.