Genome Analysis inNeurospora crassa;Cloning of Four Lociarginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) and Associated Chromosome Walking

We have cloned fourNeurospora crassagenes by complementation analysis. Cloned genes include thearginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of theNeurosporagenome is covered in these walks.     

麻疯树种子发育过程中JcOle14.3和JcOle16.6基因的表达模式研究

油质蛋白基因对种子中油体的形成至关重要,该研究通过实时荧光定量PCR,对麻疯树的两个油质蛋白基因JcOle14.3和JcOle16.6在种子不同发育时期的表达模式进行了分析。结果表明:两个基因在种子发育初期(10~30 d)表达量逐渐升高,但表达水平均较低;40 d时表达量急剧增加并达到最高,而种子发育后期(50~55 d)两个基因表达水平均逐渐降低。由此可初步推测,JcOle14.3和JcOle16.6基因的表达量可能与种子油脂积累量存在正相关。该研究结果为麻疯树油体形成机理和油质蛋白的深入研究提供了理论基础。     

赤桉ACT1和ACT2基因的克隆与生物信息学分析

为了解赤桉(Eucalyptus camaldulensis)肌动蛋白(Actin)在生长发育过程中的功能,根据赤桉幼苗转录组数据库中的肌动蛋白基因序列,从赤桉嫩叶中克隆了2条Actin基因片段,并利用RACE技术获得Actin基因的全长cDNA,分别命名为ECACT1和EC-ACT2基因。生物信息学分析表明,这两条基因的全长cDNA分别为1533 bp和1387 bp,均含有1个编码377个氨基酸的开放阅读框。经比对分析,赤桉Actin蛋白的氨基酸序列与其他植物Actin蛋白的具有较高的相似性,并且具有Actin蛋白特有的保守序列和相关特征。因此推测这两条基因对桉树的生长发育具有一定的调控作用。     

Identification of alleles at the gliadin loci Gli-U1 and Gli-Mb1 in Aegilops biuncialis Vis.

The diversity of alleles at the gliadin loci Gli-U1 and Gli-M ^b 1 was studied in the tetraploid species Aegilops biuncialis (UUM^bM^b). The collection of 41 Ae. biuncialis accessions and F₂ seeds obtained from five crosses served as the material used in this study. Gliadins were separated by acid polyacrylamide gel electrophoresis. To determine genomic affiliation (U or M^b) of components of Ae. biuncialis gliadin pattern, accessions of Ae. umbellulata and Ae. comosa were analyzed. In Ae. biuncialis accessions, 14 alleles were identified at the locus Gli-U1 and 12 alleles, at the locus Gli-M ^b 1. The results testify to a high degree of allele diversity at major gliadin-coding loci of homeologous group 1 chromosomes of Ae. biuncialis.     

TFS1: A suppressor of cdc25 mutations in Saccharomyces cerevisiae

Summary The TFS1 gene of Saccharomyces cerevisiae is a dosage-dependent suppressor of cdc25 mutations. Overexpression of TFS1 does not alleviate defects of temperature-sensitive adenylyl cyclase (cdc35) or ras2 disruption mutations. The ability of TFS1 to suppress cdc25 is allele specific: the temperature-sensitive cdc25-1 mutation is suppressed efficiently but the cdc25-5 mutation and two disruption mutations are only partially suppressed. TFS1 maps to a previously undefined locus on chromosome XII between RDN1 and CDC42. The DNA sequence of TFS1 contains a single long open reading frame encoding a 219 amino acid polypeptide that is similar in sequence to two mammalian brain proteins. Insertion and deletion mutations in TFS1 are haploviable, indicating that TFS1 is not essential for growth.     

An extragenic suppressor ofprp24-1 defines genetic interaction betweenPRP24 andPRP21 gene products ofSaccharomyces cerevisiae

The temperature-sensitiveprp24-1 mutation defines a gene product required for the first step in pre-mRNA splicing. PRP24 is probably a component of the U6 snRNP particle. We have applied genetic reversion analysis to identify proteins that interact with PRP24. Spontaneous revertants of the temperaturesensitive (ts)prp24-1 phenotype were analyzed for those that are due to extragenic suppression. We then extended our analysis to screen for suppressors that confer a distinct conditional phenotype. We have identified a temperature-sensitive extragenic suppressor, which was shown by genetic complementation analysis to be allelic toprp21-1. This suppressor,prp21-2, accumulates pre-mRNA at the non-permissive temperature, a phenotype similar to that ofprp21-1. prp21-2 completely suppresses the splicing defect and restores in vivo levels of the U6 snRNA in theprp24-1 strain. Genetic analysis of the suppressor showed thatprp21-2 is not a bypass suppressor ofprp24-1. The suppression ofprp24-1 byprp21-2 is gene specific and also allele specific with respect to both the loci. Genetic interactions with other components of the pre-spliceosome have also been studied. Our results indicate an interaction between PRP21, a component of the U2 snRNP, and PRP24, a component of the U6 snRNP. These results substantiate other data showing U2–U6 snRNA interactions.     

Instability at the y-1 locus of Chlamydomonas reinhardtii

Summary Twenty-three spontaneous yellow mutants were isolated from two stable green strains of the unicellular green alga Chlamydomonas reinhardtii. Genetic characterization indicated that 22 of 23 mutants had a mutation at the y-1 locus, and all 22 y-1 alleles were unstable. Crosses designed to follow the inheritance of instability at the y-1 locus showed that instability is caused by a single genetic factor located at the y-1 locus or very close to it.     

Oenothera mitochondrial orf454, a gene involved in cytochrome c biogenesis corresponds to orf169 and orf322 of Marchantia

We have characterized a mitochondrial gene in Oenothera, designated orf454, capable of encoding a component of the cytochrome c biogenesis system. This open reading frame is interrupted by an intron of 941 nucleotides showing high similarity to a group II intron residing in the rpl2 gene. RNA editing, which is observed at 18 cytidine positions within the orf454 reading frame, improves the similarity to protein-coding sequences in bacteria and higher plants and removes the last 16 amino acids. orf454 also shows high sequence similarity to two overlapping reading frames (orf169 and orf322) of Marchantia mitochondria. These ORFs belong to an operon-like cluster of genes in the liverwort that is not conserved in Oenothera mitochondria. However, in bacteria these reading frames are organized like the Marchantia gene cluster. It has been shown by genetical analysis in Rhodobacter capsulatus that these genes are essential for cytochrome c biogenesis. Genes of bacterial operons — ccl1 in Rhodobacter and yejR and nrfE in Escherichia coli — show high sequence similarity to the mitochondrial reading frames orf577 and orf454 of Oenothera. orf454, which we describe here, is homologous to the C-terminal region of these bacterial genes, while the previously described orf577 is homologous to the N-terminal region.     

The Arabidopsis transposable element Tag1 is widely distributed among Arabidopsis ecotypes

Tag1 is an autonomous transposable element (3.3 kb in length) first identified as an insertion in the CHL1 (NRT1) gene of Arabidopsis thaliana. Tag1 has been found in the Landsberg erecta ecotype of A. thaliana but not in Columbia or WS. In this paper, 41 additional ecotypes were examined for the presence of Tag1. Using an internal Tag1 fragment as probe, we found that DNA from 19 of the 41 ecotypes strongly hybridized to Tag1. Almost all of the Tag1-containing ecotypes had only one or two copies of Tag1 per haploid genome, as determined by Southern blot analysis. The only exception, Bf-1 from Bretagny-sur-Orge, France, had four copies. Two ecotypes, Di-G and S96, gave identical Southern blot patterns to that of Landsberg erecta and were subsequently shown to contain Tag1 at the same two positions found in Landsberg erecta (loci designated as Tag1-2 and Tag1-3). Two other ecotypes, Ag-0 and Lo-1, had a Tag1 element located at Tag1-2 but not at Tag1-3. The distance between these two loci was determined to be 0.37 cM. Analysis of DNA from two related species, A. griffithiana and A. pumila, showed that both species contain sequences that hybridize to Tag1 and that could be amplified with an oligonucleotide specific to the terminal inverted repeats of Tag1. These results show that Tag1 and related elements are present, and may be useful for insertional mutagenesis, in many A. thaliana ecotypes and several Arabidopsis species. Received: 18 August 1997 / Accepted: 9 October 1997     

Genomic distribution of retrotransposons 297, 1731, copia, mdg1 and roo in the Drosophila melanogaster species subgroup

The intragenomic distribution of five retrotransposon families (297, 1731, copia, mdg1 and roo) in the species of the melanogaster complex was studied by comparing results of the Southern blotting technique in males and females with those of in situ hybridization. The degree of structural polymorphism of each family in the different species was also investigated by restriction enzyme analysis. It was found that genomic distribution is a trait that depends on the family and species. The distribution of roo is mainly euchromatic in the four species and 1731 is heterochromatic, but the distribution of families 297, copia and mdg1 is markedly different in the melanogaster and simulans clades. These families were mainly euchromatic in D. melanogaster but heterochromatic in its sibling species. In the simulans clade most copia and mdg1 elements are located on chromosome Y. Differences in genomic distribution are unrelated with structural conservation. The relation of intragenomic distribution to phylogeny, transpositional activity and the role of the host genome are discussed.     

Tn501 insertion mutagenesis in Pseudomonas aeruginosa PAO

Summary Transposon insertion mutagenesis of the Pseudomonas aeruginosa PAO chromosome with Tn1 and Tn501 was carried out using a mutant plasmid of R68::Tn501 temperature-sensitive for replication and maintenance. This method consists of three steps. Firstly, the temperature-independent, drug-resistant clones were selected from the strain carrying this plasmid. In the temperature-indepent clones, the plasmid was integrated into the chromosome by Tn1- or Tn501-mediated cointegrate formation. Secondly, such clones were cultivated at a permissive temperature to provoke the excision of the integrated plasmid from the chromosome. Excision occurred by the reciprocal recombination between the two copies of Tn1 or Tn501 flanking the integrated plasmid, leaving one Tn1 or Tn501 insertion on the chromosome. Thirdly, the excised plasmid was cured by cultivating these isolates at a non-permissive temperature without selection for the drug resistance. Using this method, we isolated 1 Tn1-induced and 43 Tn501-induced auxotropic mutations in this organism. Genetic mapping allowed us to identify two new genes, pur-8001 and met-8003. The Tn501-induced auxotrophic mutations were distributed non-randomly among auxotrophic genes, and the reversion of the mutations by precise excision of the Tn501 insertion occurred very rarely.     

Behaviour of the transposons Tn5 and Tn7 in Xanthomonas campestris pv. campestris

Summary Xanthomonas campestris pv. campestris was tested for its ability to maintain various plasmids after they had been transferred by conjugation from Escherichia coli donors. Broad host-range plasmids belonging to incompatibility groups P and Q could be maintained but X. campestris was unable to support replication of narrow host-range ColE1, pACYC184 and pBR325 replicons. Delivery systems based on E. coli donors of suicide plasmids and on X. campestris Hfrs were used to introduce Tn7 and Tn5 into X. campestris. Tn7 insertions were recovered at high frequency while Tn5 transposed at low frequency. Three auxotrophic Tn5 insertions were isolated but transposition of Tn7 into the X. campestris genome did not generate any auxotrophs. DNA hybridization analysis showed that Tn7 had inserted into the same hot spot(s) in all cases tested.     

Influence of the ABRUPTUS/PINOID gene on the expression of homeotic mutations apetala 3-1 and pistillata-1 in Arabidopsis Thaliana (L.) Heynh.

Results of the flower structure analysis of single mutants abr, ap3-1, and pi-1 and double mutants abr ap3-1 and abr pi-1 of the Arabidopsis thaliana are presented. An increase in the expressivity of the ap3-1 and pi-1 mutations against a background of the abr mutation which break auxin transport was detected. Unlike flowers of parental mutant plants which had stamens in our experiment, stamens were absent in basal flowers of double mutants. It can be assumed that anomalies in auxin distributions in cells can break the program of the development of stamens which is triggered by the ap3-1 and pi-1 alleles remaining the residual function during the plant growing at 21–24°C.     

Dominant mutant alleles of yeast protein kinase geneCDC15 suppress thelte1 defect in termination of M phase and genetically interact withCDC14

LTE1 encodes a homolog of GDP-GTP exchange factors for the Ras superfamily and is required at low temperatures for cell cycle progression at the stage of the termination of M phase inSaccharomyces cerevisiae. We isolated extragenic suppressors which suppress the cold sensitivity oflte1 cells and confer a temperature-sensitive phenotype on cells. Cells mutant for the suppressor alone were arrested at telophase at non-permissive temperatures and the terminal phenotype was almost identical to that oflte1 cells at non-permissive temperatures. Genetic analysis revealed that the suppressor is allelic toCDC15, which encodes a protein kinase. Thecdc15 mutations thus isolated were recessive with regard to the temperature-sensitive phenotype and were dominant with respect to suppression oflte1. We isolatedCDC14 as a low-copy-number suppressor ofcdc15-rlt1.CDC14 encodes a phosphotyrosine phosphatase (PTPase) and is essential for termination of M phase. An extra copy ofCDC14 suppressed the temperature sensitivity ofcdc15-rlt1 cells, but not that ofcdc15-1 cells. In addition, some residues that are essential for the Cdc14 PTPase activity were found to be non-essential for the suppression. These results strongly indicate that Cdc14 possesses dual functions; PTPase activity is needed for one function but not for the other. We postulate that the cooperative action of Cdc14 and Cdc15 plays an essential role in the termination of M phase.     

Phytochrome-regulated EBL1 contributes to ACO1 upregulation in rice

The 1-aminocyclopropane-1-carboxylate oxidase gene (ACO1) was upregulated in rice (Oryza sativa L.) phyAphyBphyC mutants lacking any phytochrome and containing the GCC box element, a binding site for rice ethylene-responsive element binding protein 1 (OsEREBP1), in its promoter region. Since the OsEREBP1-like gene EBL1 (OsEREBP1-LIKE 1) was significantly downregulated in phyAphyBphyC mutants, EBL1 was suspected to repress ACO1 expression in wild-type plants. However, ACO1 was downregulated in EBL1 RNA interference plants, and the total length of these plants was slightly shorter than that of wild-type plants. This study shows that EBL1 is positively regulated by phytochrome B and associated with ACO1 upregulation.     

A multicopy suppressor ofnin1-1 of the yeastSaccharomyces cerevisiae is a counterpart of theDrosophila melanogaster diphenol oxidase A2 gene,Dox-A2

NIN1 is an essential gene for growth of the yeastSaccharomyces cerevisiae and was recently found to encode a component of the regulatory subunit of the 26S proteasome. Thenin1-1 mutant is temperature sensitive and its main defect is in G₁/S progression and G₂/M progression at non-permissive temperatures. One of the two multicopy suppressors ofnin1-1, SUN2 (SUppressor of Nin1-1), was found to encode a protein of 523 amino acids whose sequence is similar to those ofDrosophila melanogaster diphenol oxidase A2 and the mouse mast-cell Tum^– transplantation antigen, P91A. The C-terminal half of Sun2p was found to be functional as Sun2p at 25° C, 30° C, and 34° C but not at 37° C. The open reading frame (ORF) of theDrosophila diphenol oxidase A2 gene (Dox-A2) was obtained from a lambda phage cDNA library using the polymerase chain reaction technique. TheDox-A2 ORF driven by theTDH3 promoter complemented the phenotype of a strain deleted forsun2. ThisDox-A2-dependent strain was temperature sensitive and accumulated dumb-bell-shaped cells, with an undivided nucleus at the isthmus, after temperature upshift. This morphology is similar to that ofnin1-1 cells kept at a restrictive temperature. These results suggest thatSUN2 is a functional counterpart ofDox-A2 and that these genes play a pivotal role in the cell cycle in each organism.