Isolation and characterization of recessive sporeless mutants in the basidiomycete Coprinus cinereus

Summary Twenty-four sporeless mutants were isolated from an Amut Bmut strain (mutant in the incompatibility factors) of the basidiomycete Coprinus cinereus. All the sporeless mutations were recessive to the wild type. These mutants and a previously isolated recessive sporeless strain, N2-7 (Kanda and Ishikawa 1986) were crossed with a wildtype strain. An F1 random spore analysis indicated that sporulation deficiencies in these mutants were caused by single nuclear gene mutations. These mutations were all complementary to each other, thus twenty-five sporulation genes were identified. Five of them were linked to the A incompatibility factor. Cytological observations classified these mutants into the following four types according to the stage of the blockage: (1) meiosis stopped at meta-anaphase I; (2) meiosis was completed, but further basidial development did not occur; (3) basidial development stopped at the sterigma stage; (4) basidial development stopped at the prespore stage.     

Dominance,overdominance and epistasis in Pisum sativum L.

Summary Dominant genes are the main cause of the heterosis induced by fasciated mutants of different lines of Pisum sativum. Most of these cases were originally interpreted by different authors as examples of monogenic overdominance. Several not-closely-linked genes appear to have mutated simultaneously in most of the fasciated lines. Although fasciation itself is recessive, other mutant characters, such as lateness, increased stem length (number and length of internodes) and, in part, seed production per plant, show dominant inheritance. The latter two features are, however, to a considerable extent suppressed in the fasciated lines by unfavourable gene-interactions (epistasis). Crossing these lines with non-fasciated ones shows that the epistatic genes are recessive and the dominant genes are then no longer hindered in their action. By eliminating the epistatic genes from the genomes of fasciated lines by recombination, the heterosis phenomenon has been fixed on six independent occasions for different lines. The fasciata genes themselves were found to be the most probable cause of these cases of recessive epistasis. The question whether different kinds of fasciation affect heterosis differently is examined. Recessive epistasis and dominance explain most of the quantitative distinctions between the different hybrids. In addition, one example of heterosis between non-fasciated lines is given and the possible meaning of the overall results for plant breeding and population genetics is mentioned.     

Inheritance of resistance to watermelon mosaic virus in the cucumber line TMG-1: tissue-specific expression and relationship to zucchini yellow mosaic virus resistance

The inbred cucumber (Cucumis sativus L.) line TMG-1 is resistant to three potyviruses:zucchini yellow mosaic virus (ZYMV), watermelon mosaic virus (WMV), and the watermelon strain of papaya ringspot virus (PRSV-W). The genetics of resistance to WMV and the relationship of WMV resistance to ZYMV resistance were examined. TMG-1 was crossed with WI-2757, a susceptible inbred line. F₁, F₂ and backcross progeny populations were screened for resistance to WMV and/or ZYMV. Two independently assorting factors conferred resistance to WMV. One resistance was conferred by a single recessive gene from TMG-1 (wmv-2). The second resistance was conferred by an epistatic interaction between a second recessive gene from TMG-1 (wmv-3) and either a dominant gene from WI-2757 (Wmv-4) or a third recessive gene from TMG-1 (wmv-4) located 20–30 cM from wmv-3. The two resistances exhibited tissue-specific expression. Resistance conferred by wmv-2 was expressed in the cotyledons and throughout the plant. Resistance conferred by wmv-3 + Wmv-4 (or wmv-4) was expressed only in true leaves. The gene conferring resistance to ZYMV appeared to be the same as, or tightly linked to one of the WMV resistance genes, wmv-3.     

Medicago truncatula,a model plant for studying the molecular genetics of theRhizobium-legume symbiosis

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.     

The use of haploidy to develop plants that express several recessive traits using light-seeded canola (Brassica napus) as an example

Summary The use of haploidy to introgress recessive traits into Brassica napus canola is illustrated by describing the properties of doubled haploids obtained by microspore culture from crosses between a yellow-seeded rapeseed line (low erucic acid, high glucosinolate) and black-seeded canola. Of the 99 doubled haploid lines that were produced, 3 were yellow-seeded canola lines. This result was not significantly different than the predicted frequency of 1 in 64 for the homozygous recessive phenotype in a doubled haploid population segregating for six recessive genes. Thus, the study supports previous models of inheritance determined for yellow seededness and glucosinolate content in Brassica napus. Also, since the chances of obtaining a plant with the same characteristics in a F₂ population are 1 in 4,096, the underscore results the advantages of using haploidy to introgress recessive traits into Brassica napus canola.     

苏云金芽胞杆菌Sigma54和CcpA共同调控的基因鉴定

【目的】通过综合分析苏云金芽胞杆菌(Bacillus thuringiensis)HD73菌株Sigma54缺失突变体的转录组数据和蜡样芽胞杆菌(Bacillus cereus)ATCC 14579菌株CcpA缺失突变体的转录组数据,并进行启动子与CcpA蛋白的体外结合验证,明确Bt HD73菌株中Sigma54和CcpA共同调控的基因,丰富了对微生物的代谢调控网络的认识。【方法】以转录组测序结果为基础,通过基因同源性的比对在Bt HD73菌株中寻找受Sigma54和CcpA共同调控的基因,在这些基因中找到具有cre序列的启动子,通过凝胶阻滞验证这些启动子与CcpA蛋白的结合。【结果】Bt HD73菌株中有31个基因受Sigma54和CcpA共同调控,其中14个基因的启动子序列包含cre序列,这些启动子都可以与CcpA蛋白发生体外结合。【结论】Bt HD73菌株中有14个基因直接受CcpA的调控,同时其转录受Sigma54的控制。     

The genetics of teratocarcinoma transplantation: tumor formation in allogeneic hosts by the embryonal carcinoma cell lines F9 and PCC3

Two cultured lines of murine embryonal carcinoma, F9 and PCC3, have been grafted to a variety of allogeneic hosts. The host strains have been classified by their resistance or sensitivity to these carcinomas. Resistance seems to be immunological in nature.Allograft rejection does not correlate withH-2 haplotype, and seems to be controlled by a limited number of recessive factors, presumably histocompatibility genes. We infer that these factors have limited polymorphism in the mouse species. Recombinational analysis of strain A/He has revealed the presence of a recessive factor linked to theH-2 locus. Tumor resistance of strains C57BL/6 and AKR appears to result from the interaction of dominant or semi-dominant factors in theH-2 region with other recessive elements in the genetic background.Though F₁ hybrids between resistant mouse strains and the syngeneic strain 129 are largely tumor-sensitive, a low level of hybrid resistance to F9 has been observed and shown to be eliminated by X-irradiation.     

Second-site modifiers of the split mutation of Notch define genes involved in neurogenesis in Drosophila melanogaster

Summary We have searched for dominant modifiers, i.e., enhancers and suppressors, of the compound eye phenotype of split, a recessive viable allele of Notch. Among the spl modifiers found, we have detected mutations in loci whose functions were previously known to cooperate with Notch in embryonic neurogenesis, such as daughterless, master mind, Delta and Hairless. In addition, other spl modifier mutations have been found in loci that were not previously known to interact with Notch, such as scabrous, glass, roughened eye, and several other genes that have not yet been assigned to known loci. The phenotypes associated with mutations in some of these latter loci suggest the participation of the corresponding genes in embryonic neurogenesis. We show that in some cases the observed interactions are due to genetic haplo-insufficent expression of the genes, whereas allele-specific interactions with spl are observed in master mind and Delta alleles. From this observation, we propose a direct functional association between the proteins encoded by Notch, Delta and master mind.     

Detection of a novel spotted fever group rickettsia in <Emphasis Type="Italic">Amblyomma parvum</Emphasis> ticks (Acari: Ixodidae) from Argentina

The present study evaluated the rickettsial infection in Amblyomma parvum ticks collected in Northwestern Córdoba Province, Argentina. Each tick was subjected to DNA extraction and tested by polymerase chain reaction (PCR) targeting fragments of the rickettsial genes gltA and ompB. Nine (69.2%) out of 13 adult ticks yielded expected PCR products for the two rickettsial genes. Products from the ompB PCR were sequenced, generating DNA sequences 100% identical for the nine PCR-positive ticks. Three of these ticks were tested in another battery of PCR targeting fragments of the rickettsial genes gltA, htrA, and ompA. Products from the gltA, htrA, and ompA PCRs were sequenced generating DNA sequences 100% identical for the three PCR-positive ticks. The rickettsia detected in the A. parvum ticks was designated as Rickettsia sp. strain Argentina. Phylogenetic analyses performed with partial sequences of the rickettsial genes gltA, htrA, ompB, and ompA showed that Rickettsia sp. strain Argentina belonged to the spotted fever group, being distinct from all known Rickettsia species and genotypes available in GenBank, representing possibly a new Rickettsia species. This was the first evidence of rickettsial infection in the tick A. parvum, and the third report of rickettsial infection among the Argentinean tick fauna. The role of Rickettsia sp. strain Argentina as a human pathogen is unknown. Further studies are needed to obtain tissue-cultured isolates of Rickettsia sp. strain Argentina, in order to better characterize it and to determine its taxonomic status as a new species.     

Hydroxylation of cinnamic acids and flavonoids during biosynthesis of anthocyanins in Petunia hybrida Hort.

The effect of hydroxylation genes on the hydroxylation of intermediates of flavonoid biosynthesis in Petunia hybrida is reported. In mutants homozygous recessive, for the gene An9, dihydroflavonols accumulate. The number of hydroxyl groups in the B-ring is determined by the hydroxylation genes Htl and Hfl. A similar effect of Htl and (probably) Hfl occurs in flavanone-accumulating mutants, homozygous recessive for the gene An3. Mutants dominant for Hfl probably accumulate a 5,7,3,4,5-pentahydroxyflavanone. The mutant W43, homozygous recessive for the gene An5, is blocked in an early flavonoid biosynthesis step. It accumulates p-coumaric acid together with caffeic acid. The hydroxylation genes Htl and Hfl, however, are also homozygous recessive, which indicates that the hydroxylation of p-coumaric acid to caffeic acid or derivatives of these compounds is not controlled by Htl. The accumulation of caffeic acid was observed in all mutants investigated so far, regardless of which hydroxylation genes were dominant or recessive. We conclude that hydroxylations involved in anthocyanin biosynthesis occur at the C15 level.Deceased     

Negative effects of agrocin 84-encoding Agrobacterium plasmids on symbiotic properties of Rhizobium meliloti

Two plasmids, pAgK84::Tn5-Mob from Agrobacterium radiobacter carrying genes for the production of agrocin 84, and RP4-4 from E. coli were inserted either separately or together into a strain of Rhizobium meliloti. Each of these plasmid-containing R. meliloti transconjugants was less effective than the wild type strain in their ability to fix nitrogen in Medicago tornata. The pAgK84::Tn5-Mob-containing transconjugant was significantly less effective than that containing RP4-4. The transconjugant strains were inferior to the wild type strain in their ability to nodulate seedlings and to compete for nodulation.     

Analysis of pss genes of Rhizobium leguminosarum required for exopolysaccharide synthesis and nodulation of peas: their primary structure and their interaction with psi and other nodulation genes

Summary Strains of Rhizobium leguminosarum (R. l.) biovar viciae containing pss mutations fail to make the acidic exopolysaccharides (EPS) and are unable to nodulate peas. It was found that they also failed to nodulate Vicia hirsuta, another host of this biovar. When peas were co-inoculated with pss mutant derivatives of a strain of R.l. bv viciae containing a sym plasmid plus a cured strain lacking a sym plasmid (and which is thus Nod^-, but for different reasons) but which makes the acidic EPS, normal numbers of nodules were formed, the majority of which failed to fix nitrogen (the occasional Fix⁺ nodules were pressumably induced by strains that arose as a result of genetic exchange between cells of the two inoculants in the rhizosphere). Bacteria from the Fix^- nodules contained, exclusively, the strain lacking its sym plasmid. When pss mutant strains were co-inoculated with a Nod^- strain with a mutation in the regulatory gene nodD (which is on the sym plasmid pRL1JI), normal numbers of Fix⁺ nodules were formed, all of which were occupiced solely by the nodD mutant strain. Since a mutation in nodD abolishes activation of other nod genes required for early stages of infection, these nod genes appear to be dispensable for subsequent stages in nodule development. Recombinant plasmids, containing cloned pss genes, overcame the inhibitory effects of psi, a gene which when cloned in the plasmid vector pKT230, inhibits both EPS production and nodulation ability. Determination of the sequence of the pss DNA showed that one, or perhaps two, genes are required for correcting strains that either carry pss mutations or contain multi-copy psi. The predicted polypeptide product of one of the pss genes had a hydrophobic aminoterminal region, suggesting that it may be located in the membrane. Since the psi gene product may also be associated with the bacterial membrane, the products of psi and pss may interact with each other.     

Identification of three Zwittermicin A biosynthesis-related genes from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. <Emphasis Type="Italic">kurstaki</Emphasis> strain YBT-1520

Zwittermicin A (ZwA) is a novel, broad-spectrum linear aminopolyol antibiotic produced by some Bacillus cereus and Bacillus thuringiensis. However, only part of its biosynthesis cluster has been identified and characterized from B. cereus UW85. To better understand the biosynthesis cluster of ZwA, a bacterial artificial chromosome (BAC) library of B. thuringiensis subsp. kurstaki strain YBT-1520, a ZwA-producing strain, was constructed. Two BAC clones, 1F8 and 5E2, were obtained by PCR, which overlap the known ZwA biosynthesis cluster of B. cereus UW85. This ZwA biosynthesis cluster is at least 38.6 kb and is located on the chromosome, instead of the plasmid. Partial DNA sequencing revealed both BAC clones carry three new ZwA biosynthesis-related genes, zwa6, zwa5A and zwa5B, which were found at the corresponding location of B. cereus UW85. Putative amino acid sequences of these genes shown that ZWA6 is homologous to a typical carbamoyltransferase from Streptomyces avermitilis, while ZWA5A and ZWA5B are homologs of cysteine synthetase and ornithine cyclodeaminase which jointly synthesize 2,3-diaminopropionate in the viomycin biosynthesis pathway, respectively. The identification of these three genes further supports the hypothesized ZwA biosynthesis pathway.     

Elucidation of <Emphasis Type="Italic">veA</Emphasis>-dependent genes associated with aflatoxin and sclerotial production in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> by functional genomics

The aflatoxin-producing fungi, Aspergillus flavus and A. parasiticus, form structures called sclerotia that allow for survival under adverse conditions. Deletion of the veA gene in A. flavus and A. parasiticus blocks production of aflatoxin as well as sclerotial formation. We used microarray technology to identify genes differentially expressed in wild-type veA and veA mutant strains that could be involved in aflatoxin production and sclerotial development in A. flavus. The DNA microarray analysis revealed 684 genes whose expression changed significantly over time; 136 of these were differentially expressed between the two strains including 27 genes that demonstrated a significant difference in expression both between strains and over time. A group of 115 genes showed greater expression in the wild-type than in the veA mutant strain. We identified a subgroup of veA-dependent genes that exhibited time-dependent expression profiles similar to those of known aflatoxin biosynthetic genes or that were candidates for involvement in sclerotial production in the wild type.     

The isolation and characterization of gibberellin-deficient mutants in tomato

Summary In tomato, nine independent EMS-induced mutants representing recessive mutations at three different loci (gib-1, gib-2, and gib-3) were isolated. Six of these have an almost absolute gibberellin requirement for seed germination and elongation growth. In addition, the leaves are darker green, smaller, and changed in structure as compared to wild type. The three other mutants, which germinate without GA, are allelic to specific, nongerminating mutants and have less severe mutant characteristics. The respective loci are situated on three different chromosomes. The genes identified by these mutants control steps in gibberellin biosynthesis, as endogenous gibberellins are strongly reduced.     

Recombinant Pseudomonas putida carrying both the dsz and hcu genes can desulfurize dibenzothiophene in n-tetradecane

Pseudomonas putida IFO13696, a recombinant strain with dsz desulfurization genes, desulfurized dibenzothiophene (DBT) in water but not in n-tetradecane. By introducing into this recombinant strain the hcuABC genes that take part in the uptake of DBT in the oil phase into the cell, 82% of 1 mM DBT in n-tetradecane was degraded in 24 h by resting cells. The products of hcuABC genes thus acted in the uptake of DBT in n-tetradecane into the cells and were effective in desulfurization of DBT in the hydrocarbon phase.     

Location and organization of the dimethylphenol catabolic genes of Pseudomonas CF600

Summary The gene organization of the phenol catabolic pathway of Pseudomonas CF600 has been investigated. This strain can grow on phenol and some methylated phenols by virtue of an inducible phenol hydroxylase and meta-cleavage pathway enzymes. The genes coding for these enzymes are located on pVI150, an IncP-2 degradative mega plasmid of this strain. Twenty-three kilobases of contiguous DNA were isolated from lambda libraries constructed from strains harbouring wild type and Tn5 insertion mutants of pV1150. A 19.9 kb region of this DNA has been identified which encodes all the catabolic genes of the pathway. Using transposon mutagenesis, polypeptide analysis and expression of subfragments of DNA, the genes encoding the first four enzymatic steps of the pathway have been individually mapped and found to lie adjacent to each other. The order of these genes is the same as that for isofunctional genes of TOL plasmid pWWO and plasmid NAH7.     

Nucleotide mutations in purA gene and pur operon promoter discovered in guanosine- and inosine-producing Bacillus subtilis strains

The promoter region of the pur operon, which contains 12 genes for inosine monophosphate biosynthesis from phosphoribosylpyrophosphate, and the purA gene, encoding the adenylosuccinate synthetase, were compared among wild-type and three purine-producing Bacillus subtilis strains. A single nucleotide deletion at position 55 (relative to translation start site) in purA gene was found in a high inosine-producing strain and in a high guanosine-producing strain, which correlates with the absence of adenylosuccinate synthetase activity in these strains. Within the pur operon promoter of high guanosine-producing strain, in addition to a single nucleotide deletion in PurBox1 and a single nucleotide substitution in PurBox2, there were 4 substitutions in the flanking region of the PurBoxes and 32 nucleotide mutations in the 5′ untranslated region. These mutations may explain the purine accumulation in purine-producing strains and be helpful to the rational design of high-yield recombinant strains.     

A histidine kinase sensor protein gene is necessary for induction of low pH tolerance in Sinorhizobium sp. strain BL3

The aim of this investigation was to identify and isolate genes involved in acid tolerance from Sinorhizobium sp. strain BL3. It was hypothesized that acid tolerance of strain BL3 could be enhanced by high level expression of certain genes involved in acid tolerance, following insertion of these genes in a multiple copy plasmid. A cosmid clone library of BL3 was introduced into BL3, and the transconjugant colonies were selected at low pH. A single cosmid containing genes for acid tolerance was isolated from 40 different colonies. By transposon–insertion mutagenesis, subcloning and DNA sequencing, a gene involved in acid tolerance, actX, was identified in a 4.4-kb fragment of this cosmid. The actX mutant of BL3 showed increased acid sensitivity and was complemented by the 4.4-kb subcloned fragment. Phaseolus lathyroides seedlings inoculated with recombinant strains containing multiple copies of actX showed increased symbiotic performance at low pH. By constructing an actX::gus fusion, it was shown that actX was induced at low pH. actX encodes a putative histidine kinase sensor protein of a two-component regulatory system. The method of gene identification used in this study for isolation of actX may be applied for the isolation of other genes involved in tolerance to adverse environmental factors.