Double mutants of Phycomyces with abnormal phototropism

Summary Seven genes (madA to madG) are known which effect phototropism in Phycomyces. These genes have been partially ordered with respect to the associated stimulus-response pathway. Mutants affected in these genes serve as useful probes of photosensory transduction processes in this model system. To extend and deepen the analysis of the system, we have constructed a family of 21 double mutants in all combinations for the seven mad genes. A set of seven standard alleles was adopted for this work. The double mutants were isolated from crosses between isogenic single-mutant strains of opposite mating type. After a partial physiologic screening of the progeny, the double mutants were identified by complementation tests using single-mutant strains of known genotype. For all but three of the double mutants, the photogeotropism phenotypes were distinct from those of the respective single-mutant parentals. One triple mutant (madA madB madC) was constructed as part of this work. Various applications of the double mutants and the triple mutant are discussed. Recombination analyses were performed on the progeny from seven mad crosses to complete an earlier study. The results establish that all seven mad genes are unlinked.     

Gene Duplications and Evolution of Vertebrate Voltage-Gated Sodium Channels

Voltage-gated sodium channels underlie action potential generation in excitable tissue. To establish the evolutionary mechanisms that shaped the vertebrate sodium channel α-subunit (SCNA) gene family and their encoded Na_v1 proteins, we identified all SCNA genes in several teleost species. Molecular cloning revealed that teleosts have eight SCNA genes, compared to ten in another vertebrate lineage, mammals. Prior phylogenetic analyses have indicated that the genomes of both teleosts and tetrapods contain four monophyletic groups of SCNA genes, and that tandem duplications expanded the number of genes in two of the four mammalian groups. However, the number of genes in each group varies between teleosts and tetrapods, suggesting different evolutionary histories in the two vertebrate lineages. Our findings from phylogenetic analysis and chromosomal mapping of Danio rerio genes indicate that tandem duplications are an unlikely mechanism for generation of the extant teleost SCNA genes. Instead, analyses of other closely mapped genes in D. rerio as well as of SCNA genes from several teleost species all support the hypothesis that a whole-genome duplication was involved in expansion of the SCNA gene family in teleosts. Interestingly, despite their different evolutionary histories, mRNA analyses demonstrated a conservation of expression patterns for SCNA orthologues in teleosts and tetrapods, suggesting functional conservation. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Axel Meyer]     

Five genes at the 3' end of the Klebsiella pneumoniae pulC operon are required for pullulanase secretion

The nucleotide sequence of a 5082bp fragment of chromosomal DNA from Klebsiella pneumoniae strain UNF5023 is reported. The sequence includes the last four genes of an operon of genes specifically required for the secretion of the enzyme pullulanase. All four genes (puIL, puIM, puIN and puIO) are shown to be required for pullulanase secretion, as is a fifth gene (puIK) which extends beyond the 5′ end of the sequenced DNA. The products of the puIL, puIM, puIN and puIO genes (44kD, 18kD, 27kD and 24kD, respectively) are all predicted to have one or more hydrophobic domains typical of signal sequences and/or membrane anchors, and were all found mainly associated with the inner membranes of subfractionated cells in which the corresponding genes had been expressed from the bacteriophage T7 gene 10 promoter. The results of this study increase the number of genes which have been identified as required for pullulanase secretion to eight, in addition to genes coding for components of the general export pathway.     

A newly identified barley gene, Dhn12, encoding a YSK2 DHN, is located on chromosome 6H and has embryo-specific expression

Dehydrins are water-soluble lipid-associating proteins that accumulate during low-temperature or water-deficit conditions, and are thought to play a role in freezing- and drought-tolerance in plants. Dhn genes exist as multi-gene families in plants. Previously, we screened lambda genomic libraries of two barley cultivars in an effort to isolate all of the barley Dhn genes. We identified 11 unique Dhn genes and estimated a total of 13 Dhn genes in the barley genome. To extend the collection, we used an alternative source of clones, a 1.5×Morex barley BAC library. In this library, we found nine Dhn genes that we described previously and one new Dhn gene, Dhn12. The Dhn12 gene encodes an acidic YSK₂ dehydrin. The Dhn12 gene is located on chromosome 6H, and shows a different expression pattern from all other Dhn genes identified previously. RT-PCR results show that Dhn12 expression is embryo-specific. Dhn12 is not expressed in seedling shoots under any of the conditions tested, including non-stressed as well as dehydrated, or cold-, ABA- or NaCl-treated seedlings. Received: 6 June 1999 / Accepted: 3 November 1999     

The existence of all three ParaHox genes in the clitellate annelid, Perionyx excavatus

A ParaHox gene cluster is composed of three genes (Gsx, Xlox, and Cdx). It has been proposed that all three ParaHox genes were present in the last common ancestor to the lophotrochozoan protostomes and the deuterostomes and that gene loss event has occurred in the ecdysozoan lineage. In this paper, we report the existence of all three ParaHox genes in Perionyx excavatus, a clitellate annelid. Although orthologs of each of the three ParaHox genes were previously discovered from other lopotrochozoan taxa, this study constitutes the first reported isolation of all three ParaHox genes in the same clitellate species.Bum Joon Park and Sung-Jin Cho contributed equally to this work.     

A comprehensive study on the role of the <Emphasis Type="Italic">Yersinia pestis</Emphasis> virulence markers in an animal model of pneumonic plague

We determined the role of Yersinia pestis virulence markers in an animal model of pneumonic plague. Eleven strains of Y. pestis were characterized using PCR assays to detect the presence of known virulence genes both encoded by the three plasmids as well as chromosomal markers. The virulence of all Y. pestis strains was compared in a mouse model for pneumonic plague. The presence of all known virulence genes correlated completely with virulence in the Balb/c mouse model. Strains which lacked HmsF initially exhibited visible signs of disease whereas all other strains (except wild-type strains) did not exhibit any disease signs. Forty-eight hours post-infection, mice which had received HmsF^– strains regained body mass and were able to control infection; those infected with strains possessing a full complement of virulence genes suffered from fatal disease. The bacterial loads observed in the lung and other tissues reflected the observed clinical signs as did the cytokine changes measured in these animals. We can conclude that all known virulence genes are required for the establishment of pneumonic plague in mammalian animal models, the role of HmsF being of particular importance in disease progression.     

The cyclooxygenases

Cyclooxygenases (COXs) catalyze the rate-limiting step in the production of prostaglandins, bioactive compounds involved in processes such as fever and sensitivity to pain, and are the target of aspirin-like drugs. COX genes have been cloned from coral, tunicates and vertebrates, and in all the phyla where they are found, there are two genes encoding two COX isoenzymes; it is unclear whether these genes arose from an early single duplication event or from multiple independent duplications in evolution. The intron-exon arrangement of COX genes is completely conserved in vertebrates and mostly conserved in all species. Exon boundaries largely define the four functional domains of the encoded protein: the amino-terminal hydrophobic signal peptide, the dimerization domain, the membrane-binding domain, and the catalytic domain. The catalytic domain of each enzyme contains distinct peroxidase and cyclooxygenase active sites; COXs are classified as members of the myeloperoxidase family. All COXs are homodimers and monotopic membrane proteins (inserted into only one leaflet of the membrane), and they appear to be targeted to the lumenal membrane of the endoplasmic reticulum, where they are N-glycosylated. In mammals, the two COX genes encode a constitutive isoenzyme (COX-1) and an inducible isoenzyme (COX-2); both are of significant pharmacological importance.     

Development of PCR markers for the selection of wheat stem rust resistance genes Sr24 and Sr26 in diverse wheat germplasm

The use of major resistance genes is the most cost-effective strategy for preventing stem rust epidemics in Australian wheat crops. The long-term success of this strategy is dependent on combining resistance genes that are effective against all predominant races of the pathogen, a task greatly assisted by the use of molecular markers linked to individual resistance genes. The wheat stem rust resistance genes Sr24 and Sr26 (derived from Agropyron elongatum) and SrR and Sr31 (derived from rye) are available in wheat as segments of alien chromosome translocated to wheat chromosomes. Each of these genes provides resistance to all races of wheat stem rust currently found in Australia .We have developed robust PCR markers for Sr24 and Sr26 (this study) and SrR and Sr31 (previously reported) that are applicable across a wide selection of Australian wheat germplasm. Wheat lines have recently become available in which the size of the alien segments containing Sr26, SrR and Sr31 has been reduced. Newly developed PCR-markers can be used to identify the presence of the shorter alien segment in all cases. Assuming that these genes have different gene-for-gene specificities and that the wheat industry will discourage the use of varieties carrying single genes only, the newly developed PCR markers will facilitate the incorporation of two or more of the genes Sr24, Sr26, SrR and Sr31 into wheat lines and have the potential to provide durable control to stem rust in Australia and elsewhere.     

Quantifying bacterial population dynamics in compost using 16S rRNA gene probes

Composting provides a dynamic setting for studying ecological topics such as succession, competition, and community stability in a relatively short period of time. This study used hierarchical small sub-unit-based rRNA gene probes to quantify the change in the relative abundance of phylogenetic groups common to compost in laboratory scale reactors. Bacterial 16S rRNA gene targets accounted for only 37% of all small subunit (SSU) rRNA genes initially, but increased to a maximum of 83% of the total at 84 h. The sum of rRNA genes detected using probes specific to Pseudomonas and low-G+C Gram-positive rRNA genes represented between 16% and 87% of the total. The lack of hybridization to the taxon-specific probes was most pronounced between 36 h and 60 h, when the pH was between 4.6 and 4.8. During this period the relative abundance of taxon-specific gene targets accounted for only 17–33% of the total bacterial rRNA gene targets. Pseudomonas-type 16S rRNA genes were the most abundant of the groups measured until 72 h. Those genes had their highest relative abundance at 12 h (78% of bacterial rRNA genes; 30% of all rRNA genes), after which time their relative abundance began to decline as the temperature increased. Prior to 72 h, 16S rRNA genes from low-G+C Gram-positive bacteria (LGC-GPB) represented less than 7% of the bacterial rRNA genes. However, by 84 h the relative abundance of LGC-GPB and Bacillus rRNA genes had increased to 60% and 18% of the bacterial rRNA gene targets, respectively (50% and 15% of all rRNA genes, respectively).     

Identification and expression analysis of chitinase genes related to biotic stress resistance in <Emphasis Type="Italic">Brassica</Emphasis>

Brassica is a very important vegetable group because of its contribution to human nutrition and consequent economic benefits. However, biotic stress is a major concern for these crops and molecular biology techniques offer the most efficient of approaches to address this concern. Chitinase is an important biotic stress resistance-related gene. We identified three genes designated as Brassica chitinase like protein (BrCLP1), BrCLP2 and BrCLP3 from a full-length cDNA library of Brassica rapa cv. Osome. Sequence analysis of these genes confirmed that BrCLP1 was a class IV chitinase, and BrCLP2 and BrCLP3 were class VII chitinases. Also, these genes showed a high degree of homology with other biotic stress resistance-related plant chitinases. In expression analysis, organ-specific expression of all three genes was high except BrCLP1 in all the organs tested and BrCLP2 showed the highest expression compared to the other genes in flower buds. All these genes also showed expression during all developmental growth stages of Chinese cabbage. In addition, BrCLP1 was up-regulated with certain time of infection by Pectobacterium carotovorum subsp. carotovorum in Chinese cabbage plants during microarray expression analysis. On the other hand, expression of BrCLP2 and BrCLP3 were increased after 6 h post inoculation (hpi) but decreased from 12 hpi. All these data suggest that these three chitinase genes may be involved in plant resistance against biotic stresses.     

Detection of Variation of the R-Domain Structure of Ice Nucleation Genes in Erwinia herbicola-Group Bacteria by PCR-RFLP Analysis

The structure of ice nucleation (IN) genes was compared among 20 strains of Erwinia herbicola-group bacterium of plant- and insect-origin including E. herbicola M1 (IceE) and E. ananas IN10 (inaA) that had been previously reported. When the DNAs of N-domain or C-domain were amplified, PCR products with similar size were obtained in all strains, while the size of the PCR products from the whole genes containing the R domain varied remarkably within a range of 3.8 kb to 4.4 kb. RFLP analysis of the IN genes revealed that the size of the R-domains were varied within the region from the PvuII site to DraI site, and 20 IN genes were classified into 12 groups. Furthermore, all the strains identified as E. ananas based on six bacteriological properties were different from those of E. herbicola. These results suggest that the IN genes may be distributed only in E. ananas strains among “herbicola group bacteria.” Received: 18 March 1998 / Accepted: 28 April 1998     

The complete mitochondrial genome of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae: gene order and trn gene clusters reveal a common evolutionary course for all Sordariomycetes, while intergenic regions show variation

The mitochondrial genome (mtDNA) of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae, with a total size of 24,673 bp, was one of the smallest known mtDNAs of Pezizomycotina. It contained the 14 typical genes coding for proteins related to oxidative phosphorylation, the two rRNA genes, a single intron that harbored an intronic ORF coding for a putative ribosomal protein (rps) within the large rRNA gene (rnl), and a set of 24 tRNA genes which recognized codons for all amino acids, except proline and valine. Gene order comparison with all known mtDNAs of Sordariomycetes illustrated a highly conserved genome organization for all the protein- and rRNA-coding genes, as well as three clusters of tRNA genes. By considering all mitochondrial essential protein-coding genes as one unit a phylogenetic study of these small genomes strongly supported the common evolutionary course of Sordariomycetes (100% bootstrap support) and highlighted the advantages of analyzing small genomes (mtDNA) over single genes. In addition, comparative analysis of three intergenic regions demonstrated sequence variability that can be exploited for intra- and inter-specific identification of Metarhizium. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

十字花科多肽激素类基因RALF的同源基因克隆及进化分析

多肽激素类基因是对植物生长发育起重要作用的一类基因,RALF是其中的重要一员,而十字花科在中国蔬菜作物中是重要的一大类群。为了摸清十字花科多种蔬菜作物的RALF同源基因信息,该文根据前期研究从油菜中扩增到的多肽激素类基因RALFbn的序列设计引物,从提取的十字花科芸薹属、萝卜属、蔊菜属、山芥属的7份重要蔬菜作物的基因组DNA中分别克隆到了RALF的同源序列。结果表明:7种蔬菜作物的RALF同源基因编码区均在300 bp左右,且无内含子,编码的蛋白质由79个氨基酸组成,说明RALF在十字花科4个属内的保守性较强;对RALF同源基因在十字花科4个属中的表达分析表明,该类基因在根、茎、叶、花序轴等营养器官中不表达或弱表达,但主要在生殖器官中表达,其中在总花蕾和开放花中的表达量普遍高于嫩角果中的表达量,表明该类基因在十字花科中的生理活跃期是花发育时期。同时,构建了十字花科4个属中RALF同源基因的系统树,芸薹属的油菜、甘蓝和芥蓝形成一个分支,而茎瘤芥和分蘖芥形成另外一个分支,且与萝卜属、山芥属、蔊菜属的材料聚在一支,该基因的进化途径在一定程度上也反映出这几种作物的遗传背景关系。该研究结果丰富了RALF家族信息,增添了十字花科植物的分子进化数据。     

The Cryptochrome Gene Family in Pea Includes Two Differentially Expressed <Emphasis Type="Italic">CRY2</Emphasis> Genes

The cryptochromes are a family of blue light photoreceptors that play important roles in the control of plant development. We have characterised the cryptochrome gene family in the model legume garden pea (Pisum sativum L.). Pea contains three expressed cryptochrome genes; a single CRY1 orthologue, and two distinct CRY2 genes that we have termed CRY2a and CRY2b. Genomic southern blots indicate that there are unlikely to be more CRY genes in pea. Each of the three genes encodes a full-length CRY protein that contains all the major domains characteristic of other higher plant cryptochromes. Database searches have identified Medicago truncatula expressed sequence tags (ESTs) corresponding to all three genes, whereas only a single CRY2 is represented in EST collections from the more distantly related legumes soybean and Lotus japonicus. The proteins encoded by the pea and Medicago CRY2b genes are distinguished from other CRY2 proteins by their shorter C-terminus. Expression analyses have identified marked differences in the regulation of the three genes, with CRY2b expression in particular distinguished by high-amplitude diurnal cycling and rapid repression in seedlings transferred from darkness to blue light.     

The application of DNA marker and doubled-haploid technology for stacking multiple stem rust resistance genes in wheat

In wheat, the use of gene “pyramids” or “stacks” of major genes that confer resistance to all local strains of the fungal stem rust pathogen Puccinia graminis f. sp. tritici (Pgt) can increase durability of resistance where wheat cultivars with the single gene components are not widely deployed. Stacking two or more resistance genes becomes a breeding challenge, particularly when pathogen races that discriminate the genes are not available. The use of DNA markers and doubled-haploid technology provides a route for producing lines homozygous for multiple resistance genes. We have applied this approach to produce gene pyramids of two or more of the stem rust resistance genes Sr24 and new sources of SrR, Sr31 and Sr26 on reduced alien chromatin in the genetic backgrounds of Westonia and Pavon wheat. These genes, which are all derived from “alien” sources (SrR and Sr31 from rye, Sr24 and Sr26 from Agropyron elongatum) each provide resistance to all currently known pathotypes of Pgt in Australia, and SrR and Sr26 also provide resistance against all the variants of stem rust race Ug99 (TTKS group).     

Cloning and comparative sequence analysis of the gene coding for isopenicillin N synthase in Streptomyces

Summary The genes coding for isopenicillin N synthase (IPNS) in Streptomyces jumonjinensis and S. lipmanii were isolated from recombinant phage lambda libraries using the S. clavuligerus IPNS gene as a heterologous probe. The S. jumonjinensis IPNS gene has an open reading frame coding for 329 amino acids, identical in size to that of the previously cloned S. clavuligerus IPNS gene. A partial nucleotide sequence was also determined for the S. lipmanii IPNS gene. Comparison of the predicted amino acid sequences of all three streptomycete IPNS proteins shows that they exhibit more than 70% similarity, close to that found in comparisons among fungal IPNS proteins and significantly greater than that found, approximately 60%, between Streptomyces and fungal IPNS proteins. We conclude that procaryotic and eucaryotic IPNS genes are subgroups of a single family of microbial IPNS genes. Hybridization probes prepared from IPNS genes of the above streptomycete species were used to detect analogous genes in eight other strains that included both penicillin and cephalosporin producers and non-producers. Each producer strain responded with all three probes implying the presence of an IPNS gene. Surprisingly, several non-producer strains also responded with one or two of the probes. Our results suggest that IPNS-related genes may be more prevalent in Streptomyces than previously believed.     

Erratum

Ubiquinone (UQ), also known as coenzyme Q (CoQ), is a redox-active lipid present in all cellular membranes where it functions in a variety of cellular processes. The best known functions of UQ are to act as a mobile electron carrier in the mitochondrial respiratory chain and to serve as a lipid soluble antioxidant in cellular membranes. All eukaryotic cells synthesize their own UQ. Most of the current knowledge on the UQ biosynthetic pathway was obtained by studying Escherichia coli and Saccharomyces cerevisiae UQ-deficient mutants. The orthologues of all the genes known from yeast studies to be involved in UQ biosynthesis have subsequently been found in higher organisms. Animal mutants with different genetic defects in UQ biosynthesis display very different phenotypes, despite the fact that in all these mutants the same biosynthetic pathway is affected. This review summarizes the present knowledge of the eukaryotic biosynthesis of UQ, with focus on the biosynthetic genes identified in animals, including Caenorhabditis elegans, rodents, and humans. Moreover, we review the phenotypes of mutants in these genes and discuss the functional consequences of UQ deficiency in general.