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Complementation of the movement-deficient mutations in potato virus X: potyvirus coat protein mediates cell-to-cell trafficking of C-terminal truncation but not deletion mutant of potexvirus coat protein.

The cell-to-cell movement of the GUS-tagged potato virus X (PVX) coat protein (CP) movement-deficient mutant was restored by potyviral CPs of potato virus A (PVA) and potato virus Y (PVY) in Nicotiana benthamiana leaves in transient cobombardment experiments. Viral cell-to-cell movement of PVX CP mutant was complemented in Nicotiana tabacum cv. SR1 transgenic plants expressing PVY CP: PVX RNA and polymerase were detected in the PVX CP mutant-inoculated leaves of transgenic plants. These findings demonstrated the ability of the PVX CP-deficient mutant to move from cell to cell but not long distances in the transgenic plants and suggest that CPs of potex- and potyviruses display complementary activities in the movement process. Potyviral CP alone is not able to carry out these activities, since the mutated PVX CP is indispensable for restored movement. No trans-encapsidation between potyviral CP and PVX RNA was observed. Therefore, potyviral CP facilitates the PVX CP mutant movement by the mechanism that cannot be explained by coat protein substitution. Our data also suggest that CP functioning in cell-to-cell movement is not restricted to a simple passive role in forming virions.

Biolistics↗

The pc-1 phenotype of Chlamydomonas reinhardtii results from a deletion mutation in the nuclear gene for NADPH:protochlorophyllide oxidoreductase.

The pc-1 mutant of Chlamydomonas reinhardtii has been shown to be incapable of protochlorophyllide photoconversion in vivo and is thought to be defective in light-dependent NADPH:protochlorophyllide oxidoreductase activity. We have isolated and characterized the nuclear genes encoding this enzyme from wild-type and pc-1 mutant Chlamydomonas cells. The wild-type CRlpcr-1 gene encodes a 397 amino acid polypeptide of which the N-terminal 57 residues comprise the chloroplast transit sequence. The Chlamydomonas protochlorophyllide reductase has 66-70% identity (79-82% similarity) to the higher plant enzymes. Transcripts encoding protochlorophyllide reductase are abundant in dark-grown wild-type cells, but absent or at very low levels in cells grown in the light. Similarly, immunoreactive protochlorophyllide reductase protein is also present to a greater extent in dark- versus light-grown wild-type cells. Both pc-1 and pc-1 y-7 cells lack CRlpcr-1 mRNA and the major (36 kDa) immunodetectable form of protochlorophyllide reductase consistent with their inability to photoreduce protochlorophyllide. DNA sequence analysis revealed that the lpcr gene in pc-1 y-7 cells contains a two-nucleotide deletion within the fourth and fifth codons of the protochlorophyllide reductase precursor that causes a shift in the reading frame and results in premature termination of translation. The absence of protochlorophyllide reductase message in pc-1 and pc-1 y-7 cells is likely the consequence of this frameshift mutation in the lpcr gene. Introduction of the CRlpcr-1 gene into pc-1 y-7 cells by nuclear transformation was sufficient to restore the wild-type phenotype. Transformants contained both protochlorophyllide reductase mRNA and immunodetectable enzyme protein. These studies demonstrate that pc-1 was in fact a defect in protochlorophyllide reductase activity and provide the first in vivo molecular evidence that the lpcr gene product is essential for light-dependent protochlorophyllide reduction.

Amino Acid Sequence↗

Genetic complementation of a floral homeotic mutation, apetala3, with an Arabidopsis thaliana gene homologous to DEFICIENS of Antirrhinum majus.

Among the homeotic mutants with altered floral organs, two mutants of Arabidopsis thaliana, apetala3 and pistillata, and two mutants of Antirrhinum majus, deficiens and globosa, have a homeotic conversion of the floral organs in whorl 2 and 3, namely petals to sepals and stamens to carpels. We have isolated a homologue of the DEFICIENS gene from A. thaliana wild type and shown complete complementation of apetala3 mutation by introducing the isolated gene using Agrobacterium-mediated transformation. These results show that the APETALA3 is a homologue of DEFICIENS structurally and functionally. The 5'-upstream region of APETALA3 contains three SRE-like sequence, where MADS box-containing proteins are assumed to bind and regulate expression in tissue- and stage-specific manner.

Adaptor Proteins, Signal Transducing↗

The NADP-glutamate dehydrogenase of the cyanobacterium Synechocystis 6803: cloning, transcriptional analysis and disruption of the gdhA gene.

The gdhA gene of Synechocystis PCC 6803, which encodes an NADP-dependent glutamate dehydrogenase (NADP-GDH), has been cloned by complementation of an Escherichia coli glutamate auxotroph. This gene was found to code for a polypeptide of 428 amino acid residues, whose sequence shows high identity with those of archaebacteria (42-47%), some Gram-positive bacteria (40-44%) and mammals (37%). The minimal fragment of Synechocystis DNA required for complementation (2kb) carries the gdhA gene preceded by an open reading frame (ORF2) encoding a polypeptide of 130 amino acids. ORF2 and gdhA are co-transcribed as a 1.9 kb mRNA, but shorter transcripts including only gdhA were also detected. Two promoter regions were identified upon transcriptional fusion to the cat reporter gene of a promoter probe plasmid. Transcription from the promoter upstream of ORF2 was found to be regulated depending on the growth phase of Synechocystis, in parallel to NADP-GDH activity. This promoter is expressed in Escherichia coli too, in contrast to the second promoter, located between ORF2 and gdhA, which was silent in E. coli and did not respond to the stage of growth in Synechocystis. Disruption of the cyanobacterial gdhA gene with a chloramphenicol resistance cassette yielded a mutant strain totally lacking NADP-GDH activity, demonstrating that this gene is not essential to Synechocystis 6803 under our laboratory conditions.

Amino Acid Sequence↗

Genetic analysis of the phosphinothricin-tripeptide biosynthetic pathway of Streptomyces viridochromogenes Tü494.

Streptomyces viridochromogenes Tü494 produces the antibiotic phosphinothricyl-alanyl-alanine (Ptt). Ptt-non-producing mutants were isolated following N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or UV light treatment of spore suspensions. In co-synthesis and bioconversion experiments the mutational blocks in the biosynthetic pathway were localized. The mutant NTG1 was analysed in detail. This mutant acts as a secretor for all other mutants. From bioconversion experiments with presumptive precursors circumstantial evidence was obtained that NTG1 is mutated in a gene involved in the alanylation of N-acetyl-demethyl-phosphinothricin. Using a cosmid gene library the DNA region complementing the defective gene of mutant NTG1 was isolated on a 4-kb BamHI fragment. Subcloning experiments showed that a 3-kb BglII/BamHI fragment is sufficient for complementation of mutant NTG1.

Anti-Bacterial Agents↗

DNA amplification fingerprinting of bacteria.

We have amplified short arbitrary stretches of total bacterial DNA to produce highly characteristic and complex DNA fingerprints. This DNA amplification fingerprinting (DAF) strategy involves enzymatic amplification of DNA directed by a single arbitrary oligonucleotide primer. Amplification produces a characteristic spectrum of products that is adequately resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Although DAF is simple in concept, we found that amplification parameters must be within an optimal range for reproducibility. We establish a safe window for these parameters, which include magnesium, primer and enzyme concentration as well as cycle number. The refined procedure was used to distinguish between clinical isolates of Streptococcus uberis, Klebsiella pneumoniae, and Escherichia coli. The use of template DNA concentrations higher than 1 ng.microliters-1 and high MgCl2 levels was especially important for reproducibility when amplifying small bacterial genomes. We tested a truncated Thermus aquaticus DNA polymerase, the Stoffel fragment, and found it more tolerant of reaction conditions, more efficient in the amplification of short products, and able to produce more informative fingerprints when compared to the normal thermostable polymerase from which it was derived. Because DAF produces representative fingerprints quickly and reliably from bacteria regardless of prior genetic or biochemical knowledge, we anticipate the general use of this diagnostic tool for bacterial identification and taxonomy.

Animals↗

Dinucleotide repeat polymorphism in the proteolipoprotein (PLP) gene.

We report a dinucleotide polymorphism in the first intron of the proteolipid protein (PLP) gene with a heterozygosity frequency of 0.69 useful for molecular analysis of families with X-linked neurologic disorders characterized by dysmyelination of the central nervous system, Pelizaeus-Merzbacher Disease (PMD) and X-linked Spastic Paraplegia (SPG2).

Alleles↗

Cloning of a superoxide dismutase gene from Listeria ivanovii by functional complementation in Escherichia coli and characterization of the gene product.

A gene encoding superoxide dismutase (EC 1.15.1.1., SOD) was isolated from a plasmid library of chromosomal DNA from Listeria ivanovii by functional complementation of an SOD-negative Escherichia coli host. The nucleotide sequence of the cloned gene was determined and contained an open reading frame which codes for a protein of 202 amino acid residues (calculated molecular weight 22755 Da including the amino-terminal methionine residue). Comparison of the deduced amino acid sequence of L. ivanovii SOD with previously reported SOD amino acid sequences revealed considerable homologies with Fe- and Mn-dependent SODs. Enzymatic analyses using cell lysates and the purified recombinant enzyme indicated that this SOD is manganese-dependent. The recombinant SOD accounted for up to 30% of the total soluble protein in recombinant E. coli and protected sodA sodB mutants against the toxic effects of paraquat. Subunits of the recombinant Listeria SOD and of both E. coli SODs formed enzymatically active hybrids in vivo.

Amino Acid Sequence↗

Cloning the REC1 gene of Ustilago maydis.

The REC1 gene of Ustilago maydis plays a key role in homologous recombination and the repair of damaged DNA. In order to understand the nature and functions of the gene product, the gene has been cloned by functional complementation. A 3.8 kb cloned fragment complements the pleiotropic mitotic phenotype of different rec1 alleles. It does not complement the UV sensitivity of two other sensitive mutants. Disruption of the chromosomal copy of the 1.566 kb open reading frame within this fragment reproduces the rec1 pleiotropic phenotype. Furthermore, in diploids this disrupted reading frame is unable to complement previously characterised rec1 alleles.

Blotting, Southern↗

Identification of a negative regulator for the pathogenicity island of enterohemorrhagic Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) forms histological lesions termed attaching and effacing lesions (A/E lesions) on infected large intestine tissue. The major virulence factors involved in A/E lesions reside on a locus of enterocyte effacement (LEE), a pathogenicity island. The LEE comprises 41 specific open reading frames, of which most are organized in 5 major operons, LEE1, LEE2, LEE3, LEE4, and tir(LEE5). The expression of LEE genes is regulated in a complicated manner by environmental factors such as temperature, osmolarity, and quorum sensing. Current knowledge is that regulation is hierarchical: a pivotal positive regulator, ler, is first stimulated, which in turn activates the expression of other operons. Herein, we report on the presence of a negative regulation protein located within the LEE. L0044 is 372 bp in length and is located outside of the 5 major operons. An isogenic L0044 deletion mutant displayed loss of the repression phenotype and increased synthesis of several LEE proteins when bacteria were cultured under repressive conditions that disfavor expression of LEE proteins. Reciprocally, trans expression of L0044 suppressed the expression of the LEE. Furthermore, mRNA of ler increased as a result of deleting L0044, and disrupting ler in a L0044-deleted background reversed the loss of the repression phenotype. Thus, L0044 plays a role in regulating the expression of virulence genes in EHEC by modulating the activation of ler.

Bacterial Proteins↗

Rhodoferax antarcticus sp. nov., a moderately psychrophilic purple nonsulfur bacterium isolated from an Antarctic microbial mat.

A new species of purple nonsulfur bacteria isolated from an Antarctic microbial mat is described. The organism, designated strain ANT.BR, was mildly psychrophilic, growing optimally at 15-18 degrees C with a growth temperature range of 0-25 degrees C. Cells of strain ANT.BR were highly motile curved rods and spirals, contained bacteriochlorophyll a, and showed a multicomponent in vivo absorption spectrum. A specific phylogenetic relationship was observed between strain ANT.BR and the purple bacterium Rhodoferax fermentans FR2T, and the two organisms shared several physiological and other phenotypic properties, with the notable exception of growth temperature optimum. Tests of genomic DNA hybridization, however, showed Rfx. fermentans FR2T and strain ANT.BR to be genetically distinct bacteria. Because of its unique set of properties, especially its requirement for low growth temperatures, we propose to recognize strain ANT.BR as a new species of the genus Rhodoferax, Rhodoferax antarcticus, named for its known habitat, the Antarctic.

Antarctic Regions↗

Isolation of the putP gene of Corynebacterium glutamicum and characterization of a low-affinity uptake system for compatible solutes.

Corynebacterium glutamicum accumulates the compatible solutes proline, glycine betaine, and ectoine under conditions of high osmolality. Uptake of proline is mediated by both a high-affinity and a low-affinity secondary transport system. The low-affinity uptake system also accepts glycine betaine and ectoine as substrates. In the present study, the gene encoding the high-affinity proline uptake system PutP was isolated by heterologous complementation of Escherichia coli mutant strain WG389, which lacks the transport systems BetT, PutP, ProP, and ProU and is unable to synthesize proline and glycine betaine. This gene (putP) encodes a protein of 524 amino acids that shares identity with the proline transport systems PutP of E. coli, Staphylococcus aureus, Salmonella typhimurium, Haemophilus influenzae, and Klebsiella pneumoniae. Functional studies of PutP synthesized in E. coli mutant strain MKH13, which also lacks the transport systems for compatible solutes and is unable to synthesize glycine betaine, revealed that this carrier system is not regulated by the external osmolality on the level of activity. Km values of 7.6 mM for proline and 1.3 mM for sodium as cotransported ion were determined. Deletion of the putP gene allowed the functional characterization of another proline uptake system with low affinity.

Amino Acid Sequence↗

Isolation and characterization of ButA, a secondary glycine betaine transport system operating in Tetragenococcus halophila.

Through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, we identified a single-component glycine betaine transporter from Tetragenococcus halophila, a moderate halophilic lactic acid bacterium. DNA sequence analysis characterized the ButA protein as a member of the betaine choline carnitine transporter (BCCT) family, that includes a variety of previously characterized compatible solute transporters such as OpuD from Bacillus subtilis, EctP and BetP from Corynebacterium glutamicum, and BetL from Listeria monocytogenes. When expressed in the heterologous host E. coli, the permease is specific for glycine betaine and does not transport the other osmoprotectants previously described for T. halophila (i.e. carnitine, choline, dimethylsulfonioacetate, dimethylsulfoniopropionate, and ectoine). In E. coli, statement of ButA is mainly constitutive and maximal uptake activity may result from a weak osmotic induction. This is the first study demonstrating a role for a permease in osmoregulation, and GB uptake, of a lactic acid bacterium.

Acetoin↗

Imprinted methylation profiles for proximal mouse chromosomes 11 and 7 as revealed by methylation-sensitive representational difference analysis.

Proximal mouse Chromosome (Chr) 11 shares regions of orthology with the candidate gene region for the imprinting growth disorder Silver-Russell syndrome (SRS) on human Chr 7p. It has previously been shown that mice with two maternal or two paternal copies (duplications, Dp) of proximal Chr 11 exhibit reciprocal growth phenotypes. Those with two paternal copies show fetal and placental overgrowth, while those with two maternal copies are growth retarded. The growth retardation observed in the latter is reminiscent of the intrauterine growth restriction (IUGR) observed in SRS patients with maternal uniparental disomy for Chr 7 (mUPD7). We have carried out a methylation-sensitive representational difference analysis (Me-RDA) screen to look for regions of differential methylation (DMRs) associated with imprinted genes. For these experiments, we have used mouse embryos with uniparental duplications of Chrs 11 and 7 proximal to the breakpoint of the reciprocal translocation T(7;11)40Ad. Two previously known imprinted loci associated with paternal allele hypomethylation were recovered on proximal mouse Chr 11, U2af1-rs1 and Meg1/Grb10. These two genes map 15 cM apart, so it seems likely that they are within separate imprinted domains that do not contain additional DMRs. The known imprinted gene Peg3, located on mouse proximal Chr 7, was also detected in our screen. The finding that Peg3 was differentially methylated in embryos with uniparental inheritance of proximal Chr 7 confirms that Peg3 is located proximal to the breakpoint of T40Ad in G-band 7A2. Because GRB10 has previously been reported to be a candidate gene for SRS, we analysed 22 patients for epimutations of the GRB10 differentially methylated region that could lead to the altered expression of this gene. No such mutations were found.

Aneuploidy↗

Analogues of virus resistance genes map to QTLs for resistance to sharka disease in Prunus davidiana.

Plum pox virus (PPV), the causative agent of sharka disease in Prunoideae, is one of the most serious problems affecting stone fruit production in Europe and America. Resistance to PPV was previously described in a Prunus davidiana clone, P1908, and introduced into peach (Prunus persica) genotypes. Genetic resistance to PPV displays a complex pattern of quantitative inheritance. An analysis of quantitative trait loci (QTLs) for resistance was performed on an F1 interspecific peach population obtained from a cross between the susceptible nectarine cultivar Summergrand and P. davidiana. The hybrids were graft-inoculated with PPV in duplicate following a classical procedure. The incidence of infection was evaluated four times, over two vegetative cycles, by symptom observation and enzyme-linked immunoadsorbent assays (ELISA). Restriction of systemic downward movement of the PPV virus was also evaluated by testing the susceptible rootstocks. Using both analysis of variance and non-parametric tests, six genomic regions involved in PPV resistance were detected. Depending on the scoring data considered, between 22 and 51% of the phenotypic variance could be explained by the quantitative model. One QTL, located in the distal region of linkage group 1, maps in a genomic region that is syntenic to the location of a resistance gene previously identified in the apricot cv. Goldrich. Some QTLs appeared to be temporally specific, reflecting the environmental dependence of PPV-resistance scoring. Candidate gene fragments were amplified by PCR, isolated and mapped on the peach interspecific linkage map. We report here the co-localization of three analogues of virus resistance genes with two distinct genomic regions linked to PPV resistance in P. davidiana.

Base Sequence↗

Structure and mutation analysis of the gene encoding DNA fragmentation factor 40 (caspase-activated nuclease), a candidate neuroblastoma tumour suppressor gene.

We have characterised the DFFB gene, encoding the active subunit of the apoptotic nuclease DNA fragmentation factor (DFF40). DFFB maps to 1p36, near the imprinted putative tumour suppressor gene TP73. The DFFA gene (encoding the inhibitory DFF45 subunit) also maps to 1p36.2-36.3, and we show by FISH that DFFB lies distal to DFFA. We have also mapped a processed DFFB pseudogene to chromosome 9. DFFB itself has seven coding exons spanning 10 kb. Exhaustive mutation screening of 41 neuroblastomas and other tumours in which a 1p36 tumour suppressor gene is implicated showed no tumour-specific mutations. A coding region polymorphism was used to demonstrate uniformly biallelic expression in human fetal DFFB transcripts. Since the putative neuroblastoma tumour suppressor gene in distal 1p36 is predicted to be maternally expressed, the lack of imprinting and absence of somatic mutations in DFFB indicate that it is probably not the neuroblastoma tumour suppressor gene.

Apoptosis↗

The position of t(11;22)(q23;q11) constitutional translocation breakpoint is conserved among its carriers.

The t(11;22)(q23;q11) translocation is the most common recurrent balanced translocation described in humans. Carriers are phenotypically normal and often go undetected until diagnosis as a result of infertility investigations or following the birth of chromosomally unbalanced offspring. Efficient diagnostics of t(11;22) is important for children born to carriers of the translocation and for prenatal and pre-implantation diagnosis. The translocation breakpoint on chromosome 22 is located within a region containing low copy repeats, and this site is one of the last unfilled gaps in the sequence of this chromosome. This autosome harbors multiple other low copy repeats, which have been entirely sequenced. We report a combined sequencing and fiber FISH breakpoint characterization in five translocation carriers. From one carrier a cosmid library was constructed, and two chimeric cosmids (cos4_der11 and cos6_der22) were sequenced, which showed that strong palindromes (or inverted repeats) occur on both chromosomes. The translocation breakpoints occur at the tip of both inverted repeats. The palindrome on chromosomes 22 and 11 is composed of 852 and 166 bases, respectively. Four additional carriers were studied using fiber FISH with a resolution limit of 2 kb. Analysis of breakpoints on the DNA sequence level, or at the level of fiber FISH, indicate that they occur at the same position on both chromosomes in all five carriers. Using cos6_der22, PAC 158L19 and BAC 3009A19, we demonstrate that FISH is an attractive alternative in molecular diagnostics of t(11;22), as PCR assays are not reliable, due to the presence of numerous copies of low copy repeats.

Adult↗