Positive and negative regulation of expression of the l-sorbose (sor) operon by SorC in Klebsiella pneumoniae

Summary In Klebsiella pneumoniae the gene products involved in the degradation of the ketose l-sorbose are encoded in the sor operon. It comprises, besides structural genes for uptake and catabolism, a promoter-proximal gene sorC, encoding a protein SorC of M_r 40 kDa, for which no enzymatic function has been detected. All sor genes are coordinately expressed and inducible by l-sorbose. Polar insertions and frameshift mutations in sorC cause a pleiotropic negative effect on the expression of all other sor genes. This defect is complemented in trans by the wild-type sorC ⁺ allele for frameshift mutations, but not for polar insertions. A single promoter for all sor genes, for which SorC is the activator, thus seems to be located in front of sorC. The repressor activity of SorC was demonstrated by complementation of constitutive sorC alleles with a sorC ⁺ allele leading to inducible expression of all sor genes, including sorC, which, as visualized by the use of a series of lacZ fusions, thus autoregulates its expression, both as an activator and a repressor.     

Fine Exon–Intron Structure of the Fanconi Anemia Group A (FAA) Gene and Characterization of Two Genomic Deletions

Fanconi anemia (FA) is a genetically heterogeneous disease with at least eight genes on the basis of complementation groups (FAAtoFAH). The analysis of theFAAgene in patients suggested the existence of deletions, none of which have thus far been characterized at the genomic level. A detailed restriction map of theFAAgene with the fine localization of its 43 exons is reported in this paper. We also describe the first two genomic deletions, one of 5.0 kb and another of at least 120 kb. The former was likely the result of a recombination between relatedAlusequences. Since these interspersed repeats could generate deletions and insertions by mispairing, rearrangements of this gene are a possibility in those FA families in whichFAAmutations have not been identified.     

Molecular cloning and characterization of the dpy-20 gene of Caenorhabditis elegans

We describe the molecular analysis of the dpy20 gene in Caenorhabditis elegans. Isolation of genomic sequences was facilitated by the availability of a mutation that resulted from insertion of a Tc1 transposable element into the dpy-20 gene. The Tc1 insertion site in the m474:: Tc1 allele was identified and was found to lie within the coding region of dpy-20. Three revertants (two wild-type and one partial revertant) resulted from the excision of this Tc1 element. Genomic dpy-20 clones were isolated from a library of wild-type DNA and were found to lie just to the left of the unc-22 locus on the physical map, compatible with the position of dpy-20 on the genetic map. Cosmid DNA containing the dpy-20 gene was successfully used to rescue the mutant phenotype of animals homozygous for another dpy-20 allele, e1282ts. Sequence analysis of the putative dpy-20 homologue in Caenorhabditis briggsae was performed to confirm identification of the coding regions of the C. elegans gene and to identify conserved regulatory regions. Sequence analysis of dpy-20 revealed that it was not similar to other genes encoding known cuticle components such as collagen or cuticulin. The dpy-20 gene product, therefore, identifies a previously unknown type of protein that may be directly or indirectly involved in cuticle function. Northern blot analysis showed that dpy-20 is expressed predominantly in the second larval stage and that the mRNA is not at all abundant. Data from temperature shift studies using the temperature-sensitive allele e1282ts showed that the sensitive period also occurs at approximately the second larval stage. Therefore, expression of dpy-20 mRNA and function of the DPY-20 protein are closely linked temporally.     

Molecular organization of master mind, a neurogenic gene of Drosophila melanogaster

Summary The gene master mind (mam) is located in bands 50C23-D1 of the second chromosome of Drosophila melanogaster. mam is one of the neurogenic genes, whose function is necessary for a normal segregation of neural and epidermal lineages during embryonic development. Loss of function of any of the neurogenic genes results in a mis-routeing into neurogenesis of cells that normally would have given rise to epidermis. We describe here the molecular cloning of 198 kb of genomic DNA containing the mam gene. Ten different mam mutations (point mutants and chromosomal aberrations) have been mapped within 45 kb of the genomic walk. One of the mutations, an insertion of a P-element, was originally recovered from a dysgenic cross. Four different wild-type revertants of this mutation were characterized at the molecular level and, although modifications of the insertions were found, in no case was the transposon completely excised. An unusually high number of the repetitive opa sequence, and of an additional previously unknown element, which we have called N repeat, are scattered throughout the 45 kb where the mam mutations map. The functional significance of these repeats is unknown.     

Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc, En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.     

Mapping of the sor genes for l-sorbose degradation in the chromosome of Klebsiella pneumoniae

Summary A series of mutants was isolated in Klebsiella pneumoniae strain 1033, among them mutants unable to grown on l-sorbose. Different R' plasmids carrying the sor genes and other surrounding chromosomal genes were also isolated. Each plasmid contained the structural genes sorA for an Enzyme II of the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system, sorD for a d-glucitol 6-phosphate dehydrogenase, sorE for an l-sorbose 1-phosphate reductase, and the corresponding regulator gene sorR. These structural genes are coordinately expressed and inducible by l-sorbose. Cis-dominant and pleiotropic mutations rendering the expression of the sor genes constitutive or eliminating it were isolated. Complementation of a series of mutations in Escherichia coli K12 and K. pneumoniae by various R' and F' plasmids and by P1 transduction in K. pneumoniae located the sor genes within the following gene sequence: rbs rha pfkA metB ppc argH ilv btuB rpoB metA ace sor pgi malB uvrA. The rbs-ilv gene loci tightly linked in E. coli K12 at 84 min, are separated in the map of K. pneumoniae 1033 and located at 86 and 89 min, respectively.     

Cloning of the excC and excD genes involved in the release of periplasmic proteins by Escherichia coli K12

Summary Strains of Escherichia coli K12 carrying a tolA, tolB, lky or exc mutation located at min 16.5 on the genetic map released periplasmic proteins into the extracellular medium. Wild-type genes defined by these mutations have been cloned from E. coli genomic bank made with plasmid pBR328. Subcloning experiments and complementation studies showed that lky and exc mutations were located either in the previously described tolA and tolB genes or in the newly characterized excC and excD genes. Using minicells, excC and excD gene products were identified as proteins with a molecular mass of 19 and 21 kDa, respectively.     

Regulation of the phosphate regulon of Escherichia coli: Characterization of the promoter of the pstS gene

Summary The pstS gene belongs to the phosphate regulon whose expression is induced by phosphate starvation and regulated positively by the PhoB protein. The phosphate (pho) box is a consensus sequence shared by the regulatory regions of the genes in the pho regulon. We constructed two series of deletion mutations in a plasmid in vitro, with upstream and downstream deletions in the promoter region of pstS, which contains two pho boxes in tandem, and studied their promoter activity by connecting them with a promoterless gene for chloramphenicol acetyltransferase. Deletions extending into the upstream pho box but retaining the downstream pho box greatly reduced promoter activity, but the remaining activity was still regulated by phosphate levels in the medium and by the PhoB protein, indicating that each pho box is functional. No activity was observed in deletion mutants which lacked the remaining pho box or the-10 region. Therefore, the pstS promoter was defined to include the two pho boxes and the-10 region. The PhoB protein binding region in the pstS regulatory region was studied with the deletion plasmids by a gelmobility retardation assay. The results suggest the protein binds to each pho box on the pstS promoter. A phoB deletion mutant was constructed, and we demonstrated that expression of pstS was strictly dependent on the function of the PhoB protein.     

An Escherichia coli strain deficient for both exonuclease V and deoxycytidine triphosphate deaminase shows enhanced sensitivity to ionizing radiation

An Escherichia coli mutant lacking deoxycytidine triphosphate deaminase (Dcd) activity and an unknown function encoded by a gene designated ior exhibits sensitivity to ionizing radiation whereas dcd mutants themselves are not sensitive. A DNA fragment from an E. coli genomic library that restores the wild type level of UV and gamma ray resistance to this mutant has been cloned in the multicopy vector pBR322. Comparison of its restriction map with the physical map of the E. coli chromosome revealed complete identity to the recBD genes. ior affects ATP-dependent exonuclease activity, suggesting that it is an allele of recB. This mutation alone does not confer sensitivity to UV and gamma radiation, indicating that lack of Dcd activity is also required for expression of radiation sensitivity.     

A study of integrative transformation in Schizosaccharomyces pombe

Summary Organ-specific and constitutive expression of the Arabidosis HSP18.2 gene under normal growth conditions (22° C) was observed in transgenic A. thaliana, which carried a fusion gene composed of the promoter region of HSP18.2, one of the genes for low molecular weight heat-shock proteins in Arabidopsis, and the gene for -glucuronidase (GUS) from Escherichia coli. In order to clarify the organ-specific nature of promoter expression, various mutations that affect flower morphology were introduced into the transgenic Arabidopsis line, AHS9. The results show that the pattern of expression observed in sepals, filaments, and styles is regulated in a structure-dependent manner, and suggest that the HSP18.2 gene might have an important role in the process of differentiation of flower buds, as do several other stress-related genes.     

Mutations in genes encoding extracellular matrix proteins suppress the emb-5 gastrulation defect in Caenorhabditis elegans

The second division of the gut precursor E cells is lethally accelerated during Caenorhabditis elegans gastrulation by mutations in the emb-5 gene, which encodes a presumed nuclear protein. We have isolated suppressor mutations of the temperature-sensitive allele emb-5(hc61), screened for them among dpy and other mutations routinely used as genetic markers, and identified eight emb-5 suppressor genes. Of these eight suppressor genes, at least four encode extracellular matrix proteins, i.e., three collagens and one proteoglycan. The suppression of the emb-5 gastrulation defect seemed to require the maternal expression of the suppressors. Phenotypically, the suppressors by themselves slowed down early embryonic cell divisions and corrected the abnormal cell-division sequence of emb-5 mutant embryos. We propose an indirect stress-response mechanism to be the main cause of the suppression because: (1) none of these suppressors is specific, either to particular temperature-sensitive emb-5 alleles or to the emb-5 gene; (2) suppressible alleles of genes, reported here or elsewhere, are temperature sensitive or weak; (3) the suppression is not strong but marginal; (4) the suppression itself shows some degree of temperature dependency; and (5) none of the extracellular matrix proteins identified here is known to be expressed in oocytes or early embryos, despite the present observation that the suppression is maternal. Received: 19 August 1997 / Accepted: 11 December 1997     

Barley necrotic locus nec1 encodes the cyclic nucleotide-gated ion channel 4 homologous to the Arabidopsis HLM1

Barley homolog of the Arabidopsis necrotic (disease lesion mimic) mutant HLM1 that encodes the cyclic nucleotide-gated ion channel 4 was cloned. Barley gene was mapped genetically to the known necrotic locus nec1 and subsequent sequence analysis identified mutations in five available nec1 alleles confirming barley homolog of Arabidopsis HLM1 as the NEC1 gene. Two fast neutron (FN) induced mutants had extensive deletions in the gene, while two previously described nec1 alleles had either a STOP codon in exon 1 or a MITE insertion in intron 2 which caused alternative splicing, frame shift and production of a predicted non-functional protein. The MITE insertion was consistent with the reported spontaneous origin of the nec1 Parkland allele. The third FN mutant had a point mutation in the coding sequence which resulted in an amino acid change in the conserved predicted cyclic nucleotide-gated ion channel pore region. The expression of two pathogenesis-related genes, HvPR-1a and β-1,3-glucanase, was elevated in two FN necrotic lines. Ten other members of the barley cyclic nucleotide-gated ion channel gene family were identified and their position on barley linkage map is reported. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Intragenic recombination in the CSR1 locus of Arabidopsis

Four classes of herbicides are known to inhibit plant acetolactate synthase (ALS). In Arabidopsis, ALS is encoded by a single gene, CSR1. The dominant csr1-1 allele encodes an ALS resistant to chlorsulfuron and triazolopyrimidine sulfonamide while the dominant csr1-2 allele encodes an ALS resistant to imazapyr and pyrimidyl-oxy-benzoate. The molecular distance between the point mutations in csr1-1 and csr1-2 is 1369 bp. Here we used multiherbicide resistance as a stringent selection to measure the intragenic recombination frequency between these two point mutations. We found this frequency to be 0.008 ± 0.0028. The recombinant multiherbicide-resistant allele, csr1-4, provides an ideal marker for plant genetic transformation.     

SMB基因与拟南芥细胞脱分化

应用Affymetrix基因芯片技术研究脱分化过程的全基因组表达情况,分析拟南芥叶柄细胞在脱分化过程中的差异表达基因。结果显示:(1)脱分化叶柄有4 222个基因表现出2倍或以上的表达差异,其中1 684个基因表达上调,2 538个基因表达下调。(2)半定量RT-PCR对部分差异表达基因进行验证,进一步筛选出参与拟南芥脱分化候选基因SMB(SOMBRERO)。(3)实时PCR结果表明,野生型叶柄诱导脱分化时,SMB基因表达量随着诱导时间延长而逐渐增加。(4)SMB基因功能缺失突变体smb-3经CIM诱导很难形成愈伤组织;在激素诱导不同时间段,smb-3叶柄没有明显脱分化现象,SMB基因缺失造成拟南芥叶柄脱分化障碍。研究表明,SMB基因参与拟南芥叶柄细胞脱分化过程。     

Cytogenetical analysis of the 2B3-4-2B11 region of the X chromosome of Drosophila melanogaster

Summary Of 13 ecs mutations, which affect female fertility, as revealed by complementation analysis, 7 are chromosome rearrangements involving the br complementation group. The other six show no cytologically detectable rearrangements and behave as completely or partially noncomplementing ecs alleles. All viable combinations of these 13 mutations were characterized by partial or complete female sterility. Viable heterozygotes carrying any of these mutations and the rearrangements Df(1)sta, T(1,3)sta, Df(1)St490, previously localized distal to the ecs locus, were also sterile. Using deletions and an electrophoretic mobility variant from the Staket strain, a minor chorion gene S70 has been mapped. It had been thought this gene was located in the 2B3-5 region, and corresponded to the ecs locus. However, in the present study, this gene was shown to map in the region removed by Df(1)sta (1E1-2-2B3-4) but outside that removed by Df(1)At127 (1E1-2-2A1-2), i.e. within the 2A1-2-2B3-4 region which is distal to the ecs locus. Rearrangements and point mutations at the ecs locus that result in female sterility had no effect on synthesis of the chorion protein s70. It may therefore be suggested that the chorion protein gene is not functionally associated with the ecs locus and that sterility is caused not by disruptions of the chorion protein gene but by lesions in the ecs gene itself. Thus, an ecs product, which controlls cell sensitivity to ecdysterone is also necessary for female fertility. Data on the locations of lesions affecting female fertility indicate that at least two elements at the ecs locus are essential for this function: a cis-acting distal zone with no effect on viability and a sequence within the essential part of the ecs locus. A defect in either of these zones or their separation by chromosomal rearrangement leads to female sterility.     

Mutations affecting extracellular protease production in the filamentous fungusAspergillus nidulans

The extracellular proteases ofAspergillus nidulans are known to be regulated by carbon, nitrogen and sulphur metabolite repression. In this study, a mutant with reduced levels of extracellular protease was isolated by screening for loss of halo production on milk plates. Genetic analysis of the mutant showed that it contains a single, recessive mutation, in a gene which we have designatedxprE, located on chromosome VI. ThexprE1 mutation affected the production of extracellular proteases in response to carbon, nitrogen and, to a lesser extent, sulphur limitation. Three reversion mutations,xprF1, xprF2 andxprG1, which suppressxprE1, were characterised. BothxprF andxprG map to chromosome VII but the two genes are unlinked. ThexprF1, xprF2 andxprG1 mutants showed high levels of milk-clearing activity on medium containing milk as a carbon source but reduced growth on a number of nitrogen sources. Evidence is presented that thexprE1 andxprG1 mutations alter expression of more than one protease and affect levels of alkaline protease gene mRNA.     

Construction of pBR322-ara hybrid plasmids by in vivo recombination

Summary In vivo recombination was used to clone deletions of the araBAD-araC genes of Escherichia coli onto a hybrid pBR322-ara plasmid. Genetic and physical analysis demonstrated that the desired deletions had been recombined onto the plasmid. In addition to permitting a detailed physical analysis of various ara deletions, this procedure has generated a series of plasmid cloning vehicles that can be used to clone, by in vivo recombination, any ara point mutation located within the region covered by the deletions. Hybrid plasmids containing the cloned point mutation can be distinguished from the original cloning vehicle by genetic complementation. The desired recombinant plasmid can be easily obtained because the frequency of recombination between the plasmid ara region and the chromosomal ara region is 0.025%–3%. A plasmid containing a deletion which removes the ara controlling site region and the araC gene was used to clone two types of araBAD promoter mutations and an araCmutation by in vivo recombination. Genetic and physical analysis of these plasmids established that the mutations in question had been recombined on to the ara deletion plasmid. The application of this procedure to the ara genes and to other genetic systems is discussed.