scholarly journals Cloning, primary sequence and chromosomal localization of human FMO2, a new member of the flavin-containing mono-oxygenase family

1992 ◽  
Vol 287 (1) ◽  
pp. 261-267 ◽  
Author(s):  
C T Dolphin ◽  
E A Shephard ◽  
S Povey ◽  
R L Smith ◽  
I R Phillips
Keyword(s):  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Chromosome 1 ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Primary Sequence ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Distinct Member ◽  
Human Chromosome 1

We have previously reported the cloning of cDNAs for a flavin-containing mono-oxygenase (FMO) of man, designated FMO1 [Dolphin, Shephard, Povey, Palmer, Ziegler, Ayesh, Smith & Phillips (1991) J. Biol. Chem. 266, 12379-12385], that is the orthologue of pig and rabbit hepatic FMOs. We now describe the isolation and characterization of cDNA clones for a second human FMO, which we have designated FMO2. The polypeptide encoded by the cDNAs is 558 amino acid residues long, has a calculated M(r) of 63337, and contains putative FAD- and NADP-binding sites that align exactly with those described in other mammalian FMOs. Human FMO2 has 51-53% primary sequence identity with human FMO1, rabbit pulmonary FMO and rabbit liver FMO form 2, and thus represents a fourth, distinct, member of the mammalian FMO family. The corresponding mRNA is present in low abundance in adult human liver. Southern blot hybridization with single-exon probes demonstrated that human FMO2 and FMO1 are the products of single genes. The gene encoding FMO2 (designated FMO2) was mapped, by the polymerase chain reaction, to human chromosome 1, the same chromosome on which FMO1 is located.

Characterization of cDNA clones specific for sequences developmentally regulated during Dictyostelium discoideum spore germination

1983 ◽  
Vol 3 (11) ◽  
pp. 1943-1948
Author(s):  
L J Kelly ◽  
R Kelly ◽  
H L Ennis
Keyword(s):  
Dictyostelium Discoideum ◽  
Spore Germination ◽  
Developmental Regulation ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Molecular Weights ◽  
Multiple Copies

Spore germination in the slime mold Dictyostelium discoideum was used as a model to study the developmental regulation of protein and mRNA synthesis. Changes in the synthesis of these macromolecules occur during the transition from dormant spore to amoebae. The study of the mechanisms which regulate the quantity and quality of protein synthesis can best be accomplished with cloned genes. cDNA clones which hybridized primarily with mRNAs from only spores or germinating spores and not with growing amoebae were collected. Three such clones, denoted pLK109, pLK229, and pRK270, were isolated and had inserts of approximately 500, 1,200, and 690 base pairs, respectively. Southern blot hybridization experiments suggested that each of the genes is present in multiple copies in the D. discoideum genome. RNA blot hybridizations were performed to determine the sizes of the respective mRNAs and their developmental regulation. The mRNA that hybridized to pLK109 DNA was present predominantly in spores and at 1 h after germination but was absent in growing amoebae. Its concentration dramatically dropped at 3 h. The mRNA present in spores is apparently larger (approximately 0.5 kilobase) than in the later stages of germination (0.4 kilobase), indicating processing of the RNA during germination. The mRNA that hybridized to pLK229 DNA was approximately 1.0 kilobase and was present in very low amounts during growth. Its concentration rose until 1 h after spore germination and decreased thereafter. pRK270-specific RNA was approximately 2.7 kilobases and was found predominantly at 1 h after germination. It was present in lower concentrations at 2 and 3 h after germination and was absent in spores and amoebae. In vitro translation of mRNA selected from 1-h polyadenylated RNA which was hybridized to pLK109 or pLK229 DNA gave proteins of molecular weights consistent with the sizes of the mRNAs as determined by the RNA blot analysis.

scholarly journals Structure of active chromatin: isolation and characterization of transcriptionally active chromatin from rat liver

1997 ◽  
Vol 322 (1) ◽  
pp. 273-279 ◽  
Author(s):  
Kulbhushan TIKOO ◽  
Sunita GUPTA ◽  
Q. Anwar HAMID ◽  
Vanya SHAH ◽  
Bishwanath CHATTERJEE ◽  
...  
Keyword(s):  
Rat Liver ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Micrococcal Nuclease ◽  
Active Chromatin ◽  
Chromatin Fraction ◽  
Isolation And Characterization ◽  
Bivalent Cations ◽  
Composite Gel ◽  
A Minor

Rat liver nuclei were isolated in low-ionic-strength buffer in the absence of bi- and multi-valent cations. Digestion of these nuclei by endogenous nuclease, micrococcal nuclease and DNase I revealed that a minor chromatin fraction was preferentially digested into poly- and oligo-nucleosomes. Southern blot hybridization with various active gene probes confirmed that these chromatin fragments represent coding and 5ƀ upstream regions of transcriptionally active chromatin. Active chromatin fragments were released selectively into the medium, with inactive chromatin remaining inside the nuclei, under the above ionic conditions. The inclusion of bivalent cations during the digestion of nuclei reversed the solubility behaviour of active chromatin. Rearrangement and exchange of histone H1 between chromatin fragments was prevented by using low-salt conditions in all steps in the absence of bivalent cations. All histones, including H1, were present in stoichiometric amounts in this active chromatin fraction. Active nucleosomes showed a lower electrophoretic mobility than bulk nucleosomes in an acrylamide/agarose composite gel in the absence of Mg2+, but were selectively bound to the gel in the presence of this ion.

scholarly journals Characterization of Toxin Plasmids in Clostridium perfringens Type C Isolates

2010 ◽  
Vol 78 (11) ◽  
pp. 4860-4869 ◽  
Author(s):  
Abhijit Gurjar ◽  
Jihong Li ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Toxin Gene ◽  
Gene Encoding ◽  
Positive Type ◽  
Toxin Genes ◽  
Type C ◽  
Beta2 Toxin ◽  
Beta Toxin

ABSTRACT Clostridium perfringens type C isolates cause enteritis necroticans in humans or necrotizing enteritis and enterotoxemia in domestic animals. Type C isolates always produce alpha toxin and beta toxin but often produce additional toxins, e.g., beta2 toxin or enterotoxin. Since plasmid carriage of toxin-encoding genes has not been systematically investigated for type C isolates, the current study used Southern blot hybridization of pulsed-field gels to test whether several toxin genes are plasmid borne among a collection of type C isolates. Those analyses revealed that the surveyed type C isolates carry their beta toxin-encoding gene (cpb) on plasmids ranging in size from ∼65 to ∼110 kb. When present in these type C isolates, the beta2 toxin gene localized to plasmids distinct from the cpb plasmid. However, some enterotoxin-positive type C isolates appeared to carry their enterotoxin-encoding cpe gene on a cpb plasmid. The tpeL gene encoding the large clostridial cytotoxin was localized to the cpb plasmids of some cpe-negative type C isolates. The cpb plasmids in most surveyed isolates were found to carry both IS1151 sequences and the tcp genes, which can mediate conjugative C. perfringens plasmid transfer. A dcm gene, which is often present near C. perfringens plasmid-borne toxin genes, was identified upstream of the cpb gene in many type C isolates. Overlapping PCR analyses suggested that the toxin-encoding plasmids of the surveyed type C isolates differ from the cpe plasmids of type A isolates. These findings provide new insight into plasmids of proven or potential importance for type C virulence.

Isolation and characterization of mutants of the citrus canker pathogen Xanthomonas axonopodis pv. citri that induce a distinct pattern of disease

2000 ◽  
Vol 78 (8) ◽  
pp. 1002-1009
Author(s):  
Shu Yun Tung ◽  
Tsong Teh Kuo
Keyword(s):  
Blot Hybridization ◽  
Random Mutagenesis ◽  
Southern Blot Hybridization ◽  
Citrus Canker ◽  
Distinct Pattern ◽  
Wild Type ◽  
In Planta ◽  
Xanthomonas Axonopodis ◽  
Isolation And Characterization ◽  
Indigenous Plasmids

Random mutagenesis with the transposon Tn5tac1 in Xanthomonas axonopodis pathovar citri, the causal agent of citrus canker, generated four mutants with altered pathogenicity. These mutants were classified into three groups: (i) the nonpathogenic (NP) mutants XT10 and XT122, which did not induce any visible symptoms in the host; (ii) the WS- mutant XT27, which induced a callus-like lesion but not a watersoaked lesion; and (iii) the CL- mutant XT37, which was unable to induce a callus-like eruption but did induce the formation of a watersoaked lesion around the infection site. The NP mutants failed to grow in planta, whereas the WS- and CL- mutants showed a reduced growth rate relative to that of the wild type. Co-inoculation of leaves with the WS- and CL- mutants did not result in complementation of their respective defects. The extent of extracellular accumulation of polysaccharide, protease, and amylase activities by each of the mutants was similar to that of the wild type. The extracellular activity of polygalacturonate lyase of XT27 was reduced relative to that of the wild type and other mutants. Unlike the wild type and other mutants, XT27 also required glutamic acid for growth in culture. Southern blot hybridization revealed that each of the mutants resulted from transposon insertion at a single site; the insertion sites for XT10 and XT27 were located in the chromosome, whereas those for XT37 and XT122 were located in the indigenous plasmids. These results provide evidence that bacterial genes contribute independently to the pathogenesis of citrus canker.Key words: citrus canker, pathogenicity genes, transposon mutagenesis.

scholarly journals Isolation and characterization of a gene encoding farnesyl diphosphate synthase from \(\textit{Panax vietnamensis}\) Ha et Grushv

2021 ◽  
Vol 43 (4) ◽  
pp. 119-128
Author(s):  
Nguyen Van Giang ◽  
Luu Han Ly ◽  
Pham Le Bich Hang ◽  
Le Thi Thu Hien
Keyword(s):  
Farnesyl Diphosphate ◽  
Farnesyl Diphosphate Synthase ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
Ginsenoside Biosynthesis ◽  
Isolation And Characterization ◽  
Key Enzyme ◽  
As Stress ◽  
Root Tissues

Panax vietnamensis Ha et Grushv. is a species of the genus Panax native to Central Vietnam, containing a family of triterpene saponins named ginsenosides. This group of biomolecules possesses valuable therapeutic properties against cancer, hepatitis, diabetes, inflammation as well as stress and anxiety. Farnesyl diphosphate synthase (FPS) is a key enzyme participating in the ginsenoside biosynthesis pathway. In this study, a FPS gene from P. vietnamensis (PvFPS) was isolated and characterized. The PvFPS cDNA contained an open reading frame of 1032 bp, encoding a polypeptide chain of 342 amino acid residues. Nucleotide sequence comparison showed that FPS was highly conserved among most species, with two Aspartate-rich motifs responsible for product chain length determination strongly sustained. PvFPS was closely related to those of the same genera and order and differed from those from other kingdoms. PvFPS expression was detected at a greater level in root tissues than in leaves in all ages. Our findings provided information concerning the properties of a crucial gene in the ginsenoside biosynthesis, thus enhancing our understanding of this important pathway.

Two Nearly Identical Aromatic Compound Hydrolase Genes in a Strong Polychlorinated Biphenyl Degrader,Rhodococcus sp. Strain RHA1

1998 ◽  
Vol 64 (6) ◽  
pp. 2006-2012 ◽  
Author(s):  
Akihiro Yamada ◽  
Hidekazu Kishi ◽  
Katsumi Sugiyama ◽  
Takashi Hatta ◽  
Kanji Nakamura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Polychlorinated Biphenyl ◽  
Blot Hybridization ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Terminal Sequence ◽  
Gene Encoding ◽  
Amino Terminal ◽  
Gene Assay ◽  
Amino Terminal Sequence

ABSTRACT The two 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD) hydrolase genes,etbD1 and etbD2, were cloned from a strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, and their nucleotide sequences were determined. TheetbD2 gene was located in the vicinity of bphAgene homologs and encoded an enzyme whose amino-terminal sequence was very similar to the amino-terminal sequence of the HOHD hydrolase which was purified from RHA1. Using the etbD2 gene fragment as a probe, we cloned the etbD1 gene encoding the purified HOHD hydrolase by colony hybridization. Both genes encode a product having 274 amino acid residues and containing the nucleophile motif conserved in α/β hydrolase fold enzymes. The deduced amino acid sequences were quite similar to the amino acid sequences of the products of the single-ring aromatic hydrolase genes, such as dmpD,cumD, todF, and xylF, and not very similar to the amino acid sequences of the products of bphDgenes from PCB degraders, including RHA1. The two HOHD hydrolase genes and the RHA1 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HPDA) hydrolase gene, bphD, were expressed in Escherichia coli, and their relative enzymatic activities were examined. The product ofbphD was very specific to HPDA, and the products ofetbD1 and etbD2 were specific to HOHD. All of the gene products exhibited poor activities against themeta-cleavage product of catechol. These results agreed with the results obtained for BphD and EtbD1 hydrolases purified from RHA1. The three hydrolase genes exhibited similar induction patterns both in an RNA slot blot hybridization analysis and in a reporter gene assay when a promoter probe vector was used. They were induced by biphenyl, ethylbenzene, benzene, toluene, and ortho-xylene. Strain RCD1, an RHA1 mutant strain lacking both the bphDgene and the etbD2 gene, grew well on ethylbenzene. This result suggested that the etbD1 gene product is involved in the meta-cleavage metabolic pathway of ethylbenzene.

Pediococcus parvulus gtf Gene Encoding the GTF Glycosyltransferase and Its Application for Specific PCR Detection of β-d-Glucan–Producing Bacteria in Foods and Beverages

2006 ◽  
Vol 69 (1) ◽  
pp. 161-169 ◽  
Author(s):  
MARIA LAURA WERNING ◽  
IDOIA IBARBURU ◽  
MARIA TERESA DUEÑAS ◽  
ANA IRASTORZA ◽  
JESÚS NAVAS ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Southern Blot Hybridization ◽  
Oenococcus Oeni ◽  
Alcoholic Beverages ◽  
Gene Encoding ◽  
Specific Pcr ◽  
Genomic Locations

Exopolysaccharide production by lactic acid bacteria is beneficial in the dairy and oat-based food industries and is used to improve the texture of the fermented products. However, β-D-glucan–producing bacteria are considered spoilage microorganisms in alcoholic beverages because their secreted exopolysaccharides alter the viscosity of cider, wine, and beer, rendering them unpalatable. The plasmidic glycosyltransferase (gtf) gene of the Pediococcus parvulus 2.6 strain isolated from ropy cider has been cloned and sequenced, and its GTF product was functionally expressed in Streptococcus pneumoniae. The GTF protein, which has glycosyltransferase activity, belongs to the COG1215 membrane-bound glycosyltransferase family, and agglutination tests revealed that the enzyme enables S. pneumoniae to synthesize β-D-glucan. PCR amplification and Southern blot hybridization showed that the gtf gene is also present at different genomic locations in the β-d-glucan producers Lacto-bacillus diolivorans G77 and Oenococcus oeni I4 strains, also isolated from ropy cider. A PCR assay has been developed for the detection of exopolysaccharide-producing bacteria. Forward and reverse primers, included respectively in the coding sequences of the putative glycosyltransferase domain and the fifth trans-membrane segment of the GTF, were designed. Analysis of 76 ropy and nonropy lactic acid bacteria validated the method for specific detection of β-d-glucan homopolysaccharide producer Pediococcus, Lactobacillus, and Oenococcus strains. The limit of the assay in cider was 3 × 102 CFU/ml. This molecular method can be useful for the detection of ropy bacteria in cider before spoilage occurs, as well as for isolation of new exopolysaccharide-producing strains of industrial interest.

scholarly journals Characterization of cDNA clones specific for sequences developmentally regulated during Dictyostelium discoideum spore germination.

1983 ◽  
Vol 3 (11) ◽  
pp. 1943-1948 ◽  
Author(s):  
L J Kelly ◽  
R Kelly ◽  
H L Ennis
Keyword(s):  
Dictyostelium Discoideum ◽  
Spore Germination ◽  
Developmental Regulation ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Molecular Weights ◽  
Multiple Copies

Spore germination in the slime mold Dictyostelium discoideum was used as a model to study the developmental regulation of protein and mRNA synthesis. Changes in the synthesis of these macromolecules occur during the transition from dormant spore to amoebae. The study of the mechanisms which regulate the quantity and quality of protein synthesis can best be accomplished with cloned genes. cDNA clones which hybridized primarily with mRNAs from only spores or germinating spores and not with growing amoebae were collected. Three such clones, denoted pLK109, pLK229, and pRK270, were isolated and had inserts of approximately 500, 1,200, and 690 base pairs, respectively. Southern blot hybridization experiments suggested that each of the genes is present in multiple copies in the D. discoideum genome. RNA blot hybridizations were performed to determine the sizes of the respective mRNAs and their developmental regulation. The mRNA that hybridized to pLK109 DNA was present predominantly in spores and at 1 h after germination but was absent in growing amoebae. Its concentration dramatically dropped at 3 h. The mRNA present in spores is apparently larger (approximately 0.5 kilobase) than in the later stages of germination (0.4 kilobase), indicating processing of the RNA during germination. The mRNA that hybridized to pLK229 DNA was approximately 1.0 kilobase and was present in very low amounts during growth. Its concentration rose until 1 h after spore germination and decreased thereafter. pRK270-specific RNA was approximately 2.7 kilobases and was found predominantly at 1 h after germination. It was present in lower concentrations at 2 and 3 h after germination and was absent in spores and amoebae. In vitro translation of mRNA selected from 1-h polyadenylated RNA which was hybridized to pLK109 or pLK229 DNA gave proteins of molecular weights consistent with the sizes of the mRNAs as determined by the RNA blot analysis.

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