Sinorhizobium fredii HH103 Has a Truncated nolO Gene Due to a -1 Frameshift Mutation That Is Conserved Among Other Geographically Distant S. fredii Strains

Strain SVQ121 is a mutant derivative of Sinorhizobium fredii HH103 carrying a transposon Tn5-lacZ insertion into the nolO-coding region. Sequence analysis of the wild-type gene revealed that it is homologous to that of Rhizobium sp. NGR234, which is involved in the 3 (or 4)-O-carbamoylation of the nonreducing terminus of Nod factors. Downstream of nolO, as in Rhizobium sp. NGR234, the noeI gene responsible for methylation of the fucose moiety of Nod factors was found. SVQ121 Nod factors showed lower levels of methylation into the fucosyl residue than those of HH103, suggesting a polar effect of the transposon insertion into nolO over the noeI gene. A noeI HH103 mutant was constructed. This mutant, SVQ503, produced Nod factors devoid of methyl groups, confirming that the S. fredii noeI gene is functional. Neither the nolO nor the noeI mutation affected the ability of HH103 to nodulate several host plants, but both mutations reduced competitiveness to nodulate soybean. The Nod factors produced by strain HH103, like those of other S. fredii isolates, lack carbamoyl residues. By using specific polymerase chain reaction primers, we sequenced the nolO gene of S. fredii strains USDA192, USDA193, USDA257, and 042B(s). All the analyzed strains showed the same -1 frameshift mutation that is present in the HH103 nolO-coding region. From these results, it is concluded that, regardless of their geographical origin, S. fredii strains carry the nolO-coding region but that it is truncated by the same base-pair deletion.

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Identification of a Novel Factor V A2 Domain Tyrosine Deletion Mutation in a Patient with Factor V Deficiency and Bleeding.

Blood ◽

10.1182/blood.v104.11.4033.4033 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4033-4033

Author(s):

Carol D. Jones ◽

Fernando Negro ◽

Katherine Darnell ◽

James L. Zehnder

Keyword(s):

Amino Acids ◽

Frameshift Mutation ◽

Coagulation Factor ◽

Severe Bleeding ◽

In Vitro Mutagenesis ◽

Factor V ◽

Coding Region ◽

Base Pair Deletion ◽

A2 Domain ◽

Factor Function

Abstract The gene for coagulation Factor V (FV) is located on chromosome 1q23. FV deficiency shows an autosomal recessive mode of inheritance; heterozygotes are generally not clinically affected. The homozygous clinical phenotype occurs in approximately 1 per million individuals with variable severity of bleeding. Thus, genotype-phenotype correlations are likely to shed light on functionally important residues of FV. Here we describe a case of FV deficiency with a severe bleeding phenotype. The proband is a male infant from Argentina. His parents are unrelated. He was born healthy with no bleeding from the umbilical stump or other symptoms. He presented at eight months with a CNS hemorrhage, then suffered a second massive subdural bleed at nine months of age. Both episodes required surgical drainage and treatment with fresh frozen plasma He continues to receive prophylactic FFP infusions and has some residual neurologic impairment. The proband’s FV activity ranges from 2–14%. Two siblings are unaffected. His father’s FV activity is 50% and his mother’s is 70%. We performed DNA sequencing spanning the entire coding region of the proband’s FV gene and found two heterozygous mutations: a heterozygous single base pair deletion, del 2952T in exon 13, located in the B-domain of the FV protein, causing a frameshift mutation followed by a premature termination codon 3 amino acids downstream; and a novel 3-bp deletion in exon 10. This deletion is in-frame and results in the deletion of Y478. The del 2952T frameshift mutation was present in the father, while the del Y478 mutation was present in the mother. Y478 is in the A2 domain of FV and adjacent to another tyrosine, Y477. Evidence suggests that these tyrosine residues are important for co-factor function. Tyrosine residue sulfation has been shown to be required for full activity of the homologous co-factor, FVIII, as well as for hirudin. These sulfated tyrosines and surrounding acidic amino acids have been proposed to be important in interactions with the thrombin anion binding exosite; in the case of hirudin, sulfation of a carboxy-terminal tyrosine increases the affinity for thrombin 10-fold. The homologous tyrosines, Y718 and Y719 appear to be sulfated in FVIII. FV has been shown to be sulfated, but the precise location of the FV sulfation sites has not yet been determined. One of this patient’s FV alleles is nonfunctional due to a frameshift and a premature trancation of translation. With respect to the other allele, we hypothesize that, like FVIII, one or both of FV tyrosines 477 and 478 is sulfated, and that deletion of Y478 may result in disruption of FV co-factor function. In vitro mutagenesis and expression studies to characterize the functional consequences of the del Y478 and/or del Y477 are in progress.

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Mutation in GDP-Fucose Synthesis Genes of Sinorhizobium fredii Alters Nod Factors and Significantly Decreases Competitiveness to Nodulate Soybeans

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1999.12.3.207 ◽

1999 ◽

Vol 12 (3) ◽

pp. 207-217 ◽

Cited By ~ 46

Author(s):

Youssef Lamrabet ◽

Ramón A. Bellogín ◽

Teresa Cubo ◽

Rosario Espuny ◽

Antonio Gil ◽

...

Keyword(s):

Fatty Acyl ◽

Side Chain ◽

Broad Host Range ◽

Nod Factors ◽

Nucleotide Sugar ◽

Sinorhizobium Fredii ◽

Hindiii Fragment ◽

Transposon Insertion ◽

Nodulation Rate ◽

Soybean Nodulation

We mutagenized Sinorhizobium fredii HH103-1 with Tn5- B20 and screened about 2,000 colonies for increased β-galactosidase activity in the presence of the flavonoid naringenin. One mutant, designated SVQ287, produces lipochitooligosaccharide Nod factors (LCOs) that differ from those of the parental strain. The nonreducing N-acetylglucosamine residues of all of the LCOs of mutant SVQ287 lack fucose and 2-O-methylfucose substituents. In addition, SVQ287 synthesizes an LCO with an unusually long, C20:1 fatty acyl side chain. The transposon insertion of mutant SVQ287 lies within a 1.1-kb HindIII fragment. This and an adjacent 2.4-kb HindIII fragment were sequenced. The sequence contains the 3′ end of noeK, nodZ, and noeL (the gene interrupted by Tn5-B20), and the 5′ end of nolK, all in the same orientation. Although each of these genes has a similarly oriented counterpart on the symbiosis plasmid of the broad-host-range Rhizobium sp. strain NGR234, there are significant differences in the noeK/nodZ intergenic region. Based on amino acid sequence homology, noeL encodes GDP-D-mannose dehydratase, an enzyme involved in the synthesis of GDP-Lfucose, and nolK encodes a NAD-dependent nucleotide sugar epimerase/dehydrogenase. We show that expression of the noeL gene is under the control of NodD1 in S. fredii and is most probably mediated by the nod box that precedes nodZ. Transposon insertion into noeL has two impacts on symbiosis with Williams soybean: nodulation rate is reduced slightly and competitiveness for nodulation is decreased significantly. Mutant SVQ287 retains its ability to form nitrogen-fixing nodules on other legumes, but final nodule number is attenuated on Cajanus cajan.

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Woot, an Active Gypsy-Class Retrotransposon in the Flour Beetle, Tribolium castaneum, is Associated With a Recent Mutation

Genetics ◽

10.1093/genetics/143.1.417 ◽

1996 ◽

Vol 143 (1) ◽

pp. 417-426

Author(s):

Richard W Beeman ◽

M Scott Thomson ◽

John M Clark ◽

Marco A DeCamillis ◽

Susan J Brown ◽

...

Keyword(s):

Sequence Analysis ◽

Tribolium Castaneum ◽

Frameshift Mutation ◽

Open Reading Frames ◽

Red Flour Beetle ◽

Intron Sequence ◽

Coding Region ◽

Long Terminal Repeats ◽

Flour Beetle ◽

Reading Frames

Abstract A recently isolated, lethal mutation of the homeotic Abdominal gene of the red flour beetle Tribolium castaneum is associated with an insertion of a novel retrotransposon into an intron. Sequence analysis indicates that this retrotransposon, named Woot, is a member of the gypsy family of mobile elements. Most strains of T. castaneum appear to harbor ~25-35 copies of Woot per genome. Woot is composed of long terminal repeats of unprecedented length (3.6 kb each), flanking an internal coding region 5.0 kb in length. For most copies of Woot, the internal region includes two open reading frames (ORFs) that correspond to the gag and pol genes of previously described retrotransposons and retroviruses. The copy of Woot inserted into Abdominal bears an apparent single frameshift mutation that separates the normal second ORF into two. Woot does not appear to generate infectious virions by the criterion that no envelop gene is discernible. The association of Woot with a recent mutation suggests that this retroelement is currently transpositionally active in at least some strains.

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Construction of a single-transposon-insertion mutant in Rhizobium sp. strain TAL1145 from a double-insertion mutant

Letters in Applied Microbiology ◽

10.1111/j.1472-765x.1994.tb00927.x ◽

1994 ◽

Vol 19 (3) ◽

pp. 142-145 ◽

Cited By ~ 3

Author(s):

N. Parveen ◽

D. Borthakur

Keyword(s):

Insertion Mutant ◽

Transposon Insertion ◽

Rhizobium Sp

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RAD3 gene of Saccharomyces cerevisiae: nucleotide sequence of wild-type and mutant alleles, transcript mapping, and aspects of gene regulation

Molecular and Cellular Biology ◽

10.1128/mcb.5.1.17-26.1985 ◽

1985 ◽

Vol 5 (1) ◽

pp. 17-26

Author(s):

L Naumovski ◽

G Chu ◽

P Berg ◽

E C Friedberg

Keyword(s):

Saccharomyces Cerevisiae ◽

Nucleotide Sequence ◽

Coding Region ◽

Base Pairs ◽

S1 Nuclease ◽

Calculated Molecular Weight ◽

Base Pair Deletion ◽

S1 Nuclease Mapping ◽

Essential Function ◽

Nuclease Mapping

We determined the complete nucleotide sequence of the RAD3 gene of Saccharomyces cerevisiae. The coding region of the gene contained 2,334 base pairs that could encode a protein with a calculated molecular weight of 89,796. Analysis of RAD3 mRNA by Northern blots and by S1 nuclease mapping indicated that the transcript was approximately 2.5 kilobases and did not contain intervening sequences. Fusions between the RAD3 gene and the lac'Z gene of Escherichia coli were constructed and used to demonstrate that the RAD3 gene was not inducible by DNA damage caused by UV radiation or 4-nitroquinoline-1-oxide. Two UV-sensitive chromosomal mutant alleles of RAD3, rad3-1 and rad3-2, were rescued by gap repair of a centromeric plasmid, and their sequences were determined. The rad3-1 mutation changed a glutamic acid to lysine, and the rad3-2 mutation changed a glycine to arginine. Previous studies have shown that disruption of the RAD3 gene results in loss of an essential function and is associated with inviability of haploid cells. In the present experiments, plasmids carrying the rad3-1 and rad3-2 mutations were introduced into haploid cells containing a disrupted RAD3 gene. These plasmids expressed the essential function of RAD3 but not its DNA repair function. A 74-base-pair deletion at the 3' end of the RAD3 coding region or a fusion of this deletion to the E. coli lac'Z gene did not affect either function of RAD3.

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Homothallic switching of Saccharomyces cerevisiae mating type genes by using a donor containing a large internal deletion

Molecular and Cellular Biology ◽

10.1128/mcb.5.8.2154-2158.1985 ◽

1985 ◽

Vol 5 (8) ◽

pp. 2154-2158

Author(s):

B Weiffenbach ◽

J E Haber

Keyword(s):

Saccharomyces Cerevisiae ◽

Gene Conversion ◽

Mating Type ◽

Gene Conversion Event ◽

Conversion Event ◽

Base Pair Deletion ◽

Mating Type Genes ◽

Type Gene ◽

Mat Locus ◽

Donor Locus

Homothallic switching of the mating type genes of Saccharomyces cerevisiae occurs by a gene conversion event, replacing sequences at the expressed MAT locus with a DNA segment copied from one of two unexpressed loci, HML or HMR. The transposed Ya or Y alpha sequences are flanked by homologous regions that are believed to be essential for switching. We examined the transposition of a mating type gene (hmr alpha 1-delta 6) which contains a 150-base-pair deletion spanning the site where the HO endonuclease generates a double-stranded break in MAT that initiates the gene conversion event. Despite the fact that the ends of the cut MAT region no longer share homology with the donor hmr alpha 1-delta 6, switching of MATa or MAT alpha to mat alpha 1-delta 6 was efficient. However, there was a marked increase in the number of aberrant events, especially the formation of haploid-inviable fusions between MAT and the hmr alpha 1-delta 6 donor locus.

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Regulatory function of the Saccharomyces cerevisiae RAS C-terminus

Molecular and Cellular Biology ◽

10.1128/mcb.7.7.2309-2315.1987 ◽

1987 ◽

Vol 7 (7) ◽

pp. 2309-2315

Author(s):

M S Marshall ◽

J B Gibbs ◽

E M Scolnick ◽

I S Sigal

Keyword(s):

Saccharomyces Cerevisiae ◽

Adenylate Cyclase ◽

Attachment Site ◽

Hydrolytic Activity ◽

Regulatory Function ◽

Coding Region ◽

Activating Mutations ◽

Terminal Domain ◽

C Terminus ◽

Type Gene

Activating mutations (valine 19 or leucine 68) were introduced into the Saccharomyces cerevisiae RAS1 and RAS2 genes. In addition, a deletion was introduced into the wild-type gene and into an activated RAS2 gene, removing the segment of the coding region for the unique C-terminal domain that lies between the N-terminal 174 residues and the penultimate 8-residue membrane attachment site. At low levels of expression, a dominant activated phenotype, characterized by low glycogen levels and poor sporulation efficiency, was observed for both full-length RAS1 and RAS2 variants having impaired GTP hydrolytic activity. Lethal CDC25 mutations were bypassed by the expression of mutant RAS1 or RAS2 proteins with activating amino acid substitutions, by expression of RAS2 proteins lacking the C-terminal domain, or by normal and oncogenic mammalian Harvey ras proteins. Biochemical measurements of adenylate cyclase in membrane preparations showed that the expression of RAS2 proteins lacking the C-terminal domain can restore adenylate cyclase activity to cdc25 membranes.

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A novel 24-base pair deletion in the coding region of CCR5 in an African population

AIDS ◽

10.1097/01.aids.0000254687.86122.fa ◽

2007 ◽

Vol 21 (1) ◽

pp. 111-113 ◽

Cited By ~ 4

Author(s):

Cecile Masquelier ◽

Jean-Yeves Servais ◽

Emmanual Rusanganwa ◽

Francois Roman ◽

Emmanuel Havuga ◽

...

Keyword(s):

Base Pair ◽

African Population ◽

Coding Region ◽

Base Pair Deletion

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Molecular Evidence that the Extracellular Cutinase Pbc1 Is Required for Pathogenicity of Pyrenopeziza brassicae on Oilseed Rape

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2003.16.6.545 ◽

2003 ◽

Vol 16 (6) ◽

pp. 545-552 ◽

Cited By ~ 38

Author(s):

Donghui Li ◽

Alison M. Ashby ◽

Keith Johnstone

Keyword(s):

Oilseed Rape ◽

Single Copy ◽

Leaf Spot Disease ◽

Molecular Evidence ◽

Wild Type ◽

In Planta ◽

Coding Region ◽

Causal Organism ◽

Type Gene ◽

Culture Supernatants

Recent evidence has suggested that cutinase is required for cuticular penetration and, therefore, is essential for pathogenicity of Pyrenopeziza brassicae, the causal organism of light leaf spot disease of oilseed rape and other brassicas. In order to acquire molecular evidence for the role of cutinase in pathogenesis, the single-copy P. brassicae cutinase gene Pbc1 was disrupted by a transformation-mediated approach. Southern hybridization analysis revealed that in one mutant, NH10-1224, the disruption was due to a tandem insertion of two copies of the disruption vector into the 5′ coding region of Pbc1. In contrast to the wild type, no expression of Pbc1 was detected during in planta growth or in cutin-induced mycelium of NH10-1224 and no cutinase activity was detected in culture supernatants from NH10-1224 using pnitrophenyl butyrate as substrate. Scanning electron microscopy of Brassica napus cotyledons infected with wild-type P. brassicae confirmed that entry into the host is by direct penetration of the cuticle. In contrast, the cutinase-deficient mutant NH10-1224 failed to penetrate the cuticular layer and was unable to develop disease symptoms. This evidence is consistent with the hypothesis that Pbc1 is required for P. brassicae to penetrate the plant cuticle. Demonstration that complementation of NH10-1224 with the Pbc1 wild-type gene restores both cutinase activity and pathogenicity will be required to definitively establish that cutinase is required for successful pathogenesis of brassicas by P. brassicae.

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