Restriction Digest Patterns Research Articles

Schistosoma mansoni tropomyosin: cDNA characterization, sequence, expression, and gene product localization

We have defined the polypeptide pattern of 3-hr Schistosoma mansoni schistosomula on nonequilibrium two-dimensional gels (NEPHGE). An acidic group of polypeptides with a molecular weight of about 40 kDa and a p I value of around 5.0 (numbered 48/59/53) were identified as antigens on Western blots probed with chronic human infection sera or vaccinated mouse sera. Polypeptides 48/49/53 from silver-stained NEPHGE gels produced antisera that were specific as demonstrated by Western blot analysis and immunoprecipitations of in vitro translation products. A cDNA clone (clone 1) from a S. mansoni adult worm pBR322 library was isolated by using cDNA probes made from size-fractionated mRNA and defined as encoding polypeptide 49 by hybridization selection of the mRNA which was in vitro translated and immunoprecipitated with specific mouse antiserum. A λ gt 11 expression clone which contained an insert close to the full length mRNA was isolated from a S. mansoni cercariae library. The complete sequence of the mRNA was determined by sequencing the insert of this clone as well as primer extension of total RNA. The only open reading frame coding for 284 amino acids in the 1316 nucleotide sequence showed a 44.76 to 55.44% homology with the amino acid sequences of 18 different tropomyosins from various species. Computer-predicted secondary structure of schistosome tropomyosin was mainly α-helix which was very similar to other tropomyosins. Northern analysis showed the mRNA to be about 1.5 kb in size and detectable at much higher levels in the adult worm stage as compared to the cercariae and the egg stages. Western blot analysis likewise showed that greater amounts of tropomyosin were detected in extracts from adult worm stage as compared to extracts from cercariae and egg stages. Immunocytochemical analysis shows that tropomyosin is strongly associated with the tegument of adult worms. The restriction digestion pattern given by genomic Southern analysis suggests the existence of introns and/or multiple gene copies. Thus polypeptide 49, an immunodominant antigen, represents schistosome tropomyosin.

Consistent oncogene methylation changes in epithelial cells chemically transformed in vitro

We have examined the restriction digest patterns of CCGG sequences in Ki- ras, Ha- ras, and c- myc oncogenes in rat tracheal epithelial cells transformed in vitro by 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene/12-O-tetradecanoylphorbol-13-acetate (TPA), or TPA alone. Oncogenes c- myc and Ha- ras in transformed cell lines, compared to normal rat tracheal epithelial cells and untreated primary cultures, had altered Hpa II restriction patterns as demonstrated by hybridizing bands of different molecular weight, or loss of bands. Ki- ras was hypermethylated in all cell derivations, including normal cells. These molecular alterations have not previously been reported for epithelial cells transformed in vitro by polycyclic hydrocarbons and tumor promoters, and suggest common mechanisms of action for agents with diverse molecular targets.

Zymomonas mobilis CP4: a clarification of strains via plasmid profiles

There has been confusion regarding the nomenclature of cultures of Zymomonas mobilis CP4 (Gonçalves de Lima et al., 1970). Thus five cultures from different laboratories but originally received from a single source (Recife, Brazil) were compared by analysis of their plasmid profiles and the restriction digest patterns of the plasmids. Four cultures of Z. mobilis CP4 showed identical plasmid profiles and restriction digest patterns. One culture of CP4 (ZM4, ATCC 31821), cultured in P.L. Rogers' laboratory in Australia, and cultures derived from it and deposited in connection with recent patents, strain ZM401 (ATCC 31822) and strain ZM481 (ATCC 31823), differed from the first four cultures with respect to both plasmid profiles and restriction digest pattern. The reason for the plasmid differences between the two groups of cultures is unclear, but may have arisen in the original distribution, by natural selection while in continued culture or perhaps by selection of a minor strain in the original culture. In order to clarify the status of the two variant groups, the cultures that possess the same plasmid profile as the CP4 culture currently being distributed from the Recife culture collection are now designated CP4 (and derivatives). The cultures that possess the plasmid profile of the Rogers' CP4 (ZM4) are now designated by the Rogers' laboratory notation as ZM4 (and derivatives) and prior citation to CP4 removed from their nomenclature.

Characterisation of methicillin-resistant Staphylococcus aureus isolates by restriction endonuclease digestion of chromosomal DNA.

Clinical isolates of Staphylococcus aureus from the London Hospital were characterised by genetic analysis of antibiotic-resistance determinants and by restriction endonuclease digestion of chromosomal DNA and compared with isolates from elsewhere in the UK. Restriction enzyme digestion of chromosomal DNA confirmed that a single strain of methicillin-resistant S. aureus (MRSA) persists at the London Hospital, although its antibiotic-resistance profile and plasmid carriage are not constant. Methicillin-sensitive isolates, on the other hand, each had readily distinguishable and unique DNA restriction patterns. The DNA restriction digest pattern of the London Hospital MRSA isolates was identical to that of "epidemic" (E) MRSA isolates from the Thames regions. By contrast, other MRSA isolates had DNA restriction patterns which differed from those of EMRSA isolates and from each other. These results confirm the discriminatory value of restriction pattern analysis as a typing method.

Plasmids in carboxydotrophic bacteria: physical and restriction analysis

Twenty species and strains of aerobic CO-oxidizing bacteria were screened for the occurrence of plasmids. Six of them harbored plasmids between 45 and 558kb. Megaplasmids of 428 and 558 kb were resolved in Alcaligenes carboxydus. Restriction digest patterns of plasmids from different carboxydotrophic bacteria were dissimilar. However, the patterns obtained with the plasmids from the strains OM5, OM4 and OM2 of Pseudomonas carboxydovorans were very much the same. The nine cured mutants of P. carboxydovorans OM5, as well as the deletion mutant OM5-29, could not grow chemolithotrophically with CO or H2 plus CO2, as they were devoid of CO dehydrogenase, hydrogenase and ribulose bisphosphate carboxylase. The deletion mutant OM5-24 retained the ability to grow with CO. It could not grow with H2 plus CO2 and was devoid of H2ase. The data suggest the residence of structural and/or regulatory genes of CODH, H2ase and RuBPCx on plasmid pHCG3 of P. carboxydovorans.

Novel densitometric method for endonuclease analysis of Escherichia coli DNA samples containing multiple plasmids.

We developed a novel method whereby the digestion pattern of each plasmid was distinguished in a sample of endonuclease-digested plasmid DNAs which contained multiple plasmids extracted from a bacterial strain. This method consisted of two procedures. (i) The concentration ratio of each undigested plasmid DNA and of each digested DNA fragment was calculated on the basis of densitometric scanning of an electrophoretogram, and the concentration ratio was then compared with the theoretical concentration ratio to determine from which plasmid each fragment was generated. (ii) The second procedure involved rapid visual identification with a scanning graph. We thus analyzed five strains of Escherichia coli that harbored several cryptic plasmids. The two procedures made it possible to analyze the restriction digestion pattern of each plasmid without the need to isolate the individual plasmids, except in the case of certain fragments. Even when the digested patterns of these exceptional fragments could not be distinguished completely, however, our method had the advantage that peak patterns of plasmids could be compared visually among different bacterial strains.

Restriction Digest Patterns of Total DNA from Different Races of Fusarium oxysporum f. sp. pisi– an Improved Method for Race Classification

AbstractSeven isolates of Fusarium oxysporum f. sp. pisi were allocated to either race 2 or race 6 by their ability or inability to cause disease on the six host differentials New Season, New Era, Dark Skin Perfection, WSU 28, WSU 23 and Little Marvel. The isolates showed similar colony morphology on various solid media and their growth rates were inhibited to a similar extent by nystatin, cycloheximide and trichodermin when these compounds were included in potato dextrose agar. Total DNA waspurified from each isolate, digested separately with the restriction endonucleases EcoRI, EcoRV, Pstl, BamHl, SmaI and PvuII and the resulting DNA fragments separated by agarose gel electrophoresis. The patterns obtained were indicative of the presenceof repetitive DNA sequences. Moreover, the patterns obtained for the five race 2 isolates were identical for a given enzyme as were the patterns for the two race 6 isolates. Hence classification of isolates into races by their restrictive enzyme digestion, patterns coincided with that according to pathotypes and could be an improved, more reliable and reproducible method of race classification. An eighth isolate, originally classified as race 1, had lost its ability to cause disease on all six host differentials. The restriction digest patterns of its DNA were different from those of the DNA from both the race 2 and the race 6 isolates.

Bradyrhizobium japonicum Serocluster 123 and Diversity among Member Isolates

Diversity was examined within a group of 79 isolates of Bradyrhizobium japonicum reactive to fluorescent antibodies (FAs) prepared against B. japonicum USDA 123. Analyses were by means of cross-adsorbed FAs, bacteriophage typing, and endonuclease restriction digest patterns. Serogroups 127 and 129 shared antigenic determinants with serogroup 123 but not with each other. Bacteriophage and DNA digest patterns reflected more common features between serogroups 123 and 127 than between 123 and 129. Serogroups 129 and 122 showed FA cross-reactivity. The term serocluster was proposed to reflect interrelationships observed among the serogroups.

Molecular epidemiology of non-O1 Vibrio cholerae and Vibrio mimicus in the U.S. Gulf Coast region.

Ten toxigenic Vibrio cholerae non-O1 and V. mimicus strains isolated from clinical and environmental sources in the U.S. Gulf Coast region were examined for genetic relatedness. Restriction digest patterns of chromosomal DNA and Southern blot analysis with a cholera toxin gene probe revealed that the strains exhibited greater genetic divergence than the highly conserved V. cholerae O1 strains isolated from clinical and sewage samples in this region.

Characterization, molecular cloning, and physical mapping of the shope fibroma virus genome

Five strains of Shope fibroma virus (SFV), a strain of rabbit myxoma virus, and a strain of vaccinia virus were compared by restriction endonuclease digestion of their viral DNAs. Restriction digest patterns revealed that SFV and rabbit myxoma, both members of the Leporipoxvirus genus, were distinct from vaccinia, an Orthopoxvirus. All strains of SFV examined had a high degree of nucleotide sequence homology as shown by conservation of restriction sites within their genomes. However, restriction patterns of SFV and myxoma were quite different from one another suggesting that the genomes from these two viruse of the Leporipoxvirus genus do not share a large, highly conserved region of homology as do the viruses belonging to the Orthopoxvirus genus. Restriction mapping identified inverted terminal repeats of approximately 12 kb in length. Restriction fragments representing all but 400 bp of the termini were cloned in plasmid vectors.

Plasmids in methanotrophic bacteria: isolation, characterization and DNA hybridization analysis.

Ten strains of obligate methanotrophs were screened for the presence of plasmid DNA using a variety of methods. Plasmids were detected in all strains except Methylococcus capsulatus Bath. No significant similarity between plasmids was observed with respect to size or restriction digest patterns except for three strains of Methylosinus trichosporium, which appeared to contain the same three plasmids. Nitrocellulose filter hybridization revealed that the plasmid DNA from the M. trichosporium strains shared a small region of homology with the plasmid DNA from Methylosinus sporium 5. All of the plasmids remain cryptic. As the first step in characterization, a restriction digest map of the 55 kb plasmid found in Methylomonas albus BG8 was constructed.

Mutations of the Adh1 gene in maize following infection with barley stripe mosaic virus.

Mutations at the Adh1 locus in maize were selected from plants infected with barley stripe mosaic virus (BSMV). Pollen from the infected inbred line 1s2p, which is homozygous for Adh1-S (abbreviated S), Adh2-P, c and r was treated with allyl alcohol and applied to silks of a tester stock homozygous for Adh1-F, Adh2-N, C and R. From these pollinations 356 kernels arose on the F1 ears. Of these eight showed no activity of the S allele in scutellar samples while two exhibited low levels. Five of the putative mutant kernels germinated and two of these contained the contamination markers Adh2-P, c and r. The newly arisen mutations were designated S5446 and S5453. S5453 exhibited an abnormally low level of ADH activity in the F1 scutellum. In the F2 generation the mutant reverted at a high frequency with only about 5% of the S5453 alleles expressing low levels. DNA blotting and hybridization analyses showed no alterations in the restriction patterns of S5453 when compared to the progenitor S allele. S5446 which exhibited no ADH activity in the F1 scutellum is unstable in the pollen; reversion frequencies approaching 10(-2) were observed in samples from some plants. Restriction digestion patterns of DNA from this mutant revealed the presence of a 3.3 kb insertion at Adh. The insert does not appear to contain sequences homologous to the BSMV genome but rigorous analyses remain to be carried out. It is hypothesized that BSMV infection may mobilize endogenous but dormant transposable elements in maize.

Gene amplification in the lac region of E. coli

We have characterized strains of E. coli in which the lac region, together with varying amounts of surrounding DNA, is amplified 40 to 200 fold. The amplification events involve regions of 7 to 37 kb and result in a tandem array of repeated units. Restriction digest patterns of DNA from over 100 independent strains reveal that the amplified units are different in each case. Mechanisms of gene duplication and amplification, and the relationship of gene amplification in bacteria to that in eucaryotic cells, are considered.

Isolation of human cytomegalovirus DNA from infected cells.

Human cytomegalovirus (HCMV) DNA was extracted from infected human embryo fibroblast cultures using the Hirt, Triton-NaCl, and total extraction methods. The Hirt method gave a maximum yield of 60% and was 5- to 10-fold more efficient than the Triton-NaCl method for the extraction of HCMV DNA. However, both methods gave comparable yields ranging from 70 to 75% for the extraction of herpes simplex virus type 1 (HSV1) DNA from infected cells. The Hirt-extracted HCMV DNA contained from 5 to 10% contaminating mitochondrial DNA which could be removed after centrifugation in CsCl-ethidium bromide density gradients. The Hirt-extracted HCMV DNA was analyzed by velocity sedimentation in sucrose gradients and was found to sediment similar to HSV1 55S DNA. Additionally, the Hirt-extracted HCMV DNA was shown to be similar to virion-extracted HCMV DNA in plaquing efficiency and by HindIII restriction digest patterns.

A DNA polymorphism, consistent with gene duplication, correlates with high renin levels in the mouse submaxillary gland

The renin regulatory locus ( Rnr) is a genetic element governing mouse submaxillary gland (SMG) renin levels. A 45,000 dalton polypeptide detectable after in vitro translation of mouse SMG mRNA has been identified by genetic and physical criteria as SMG renin. A cDNA recombinant clone specific for SMG renin has been isolated and used to demonstrate that the previously described genetic regulation of SMG renin levels is manifest at the level of renin mRNA concentration. The renin cDNA clone has also been used in Southern blot analyses to study the organization of homologous DNA sequences in strains carrying different alleles at the Rnr locus. Restriction digest patterns of high renin strains ( Rnr s ) are characteristically distinct from patterns observed for low renin strains ( Rnr b ) and are suggestive of a structural gene duplication at the chromosome 1 locus in high renin strains. However, gene dosage cannot account for the increased levels in high renin strains, since SMG renin levels in Rnr s and those in Rnr b may differ up to 100-fold.

DNA restriction endonuclease analysis for localization of human beta- and delta-globin genes on chromosome 11.

DNA from a clone of a mouse-human hybrid that retained a human chromosome consisting of the major part of chromosome 11 and region q25-26-qter of the X chromosome was digested with various restriction endonucleases, subjected to electrophoresis in agarose gels, and transferred to nitrocellulose filters. The restriction digest pattern of the clone, when hybridized with a 32P-labeled plasmid fragment containing human beta-globin gene sequences, was a composite of the normal human and mouse (A9) patterns. When back-selected in 6-thioguanine to eliminate the 11 translocation chromosome, the hybrid cells showed only the A9 restriction pattern. These results substantiate the localization of beta- and delta-globin genes to human chromosome 11 and exclude the region 11q23-qter as the site.

Restriction enzyme analysis of mitochondrial DNAs of petite mutants of yeast: Classification of petites, and deletion mapping of mitochondrial genes

We have analyzed the restriction digest patterns of the mitochondrial DNA from 41 cytoplasmic petite strains of Saccharomyces cerevisiae, that have been extensively characterized with respect to genetic markers. Each mitochondrial DNA was digested with seven restriction endonucleases (EcoRI, HPaI, HindIII, BamHI, HhaI, SalI, and PstI) which together make 41 cuts in grande mitochondrial DNA and for which we have derived fragment maps. The petite mitochondrial DNAs were also analyzed with HpaII, HaeIII, and AluI, each of which makes more than 80 cleavages in grande mitochondrial DNA. On the basis of the restriction patterns observed (i.e., only one fragment migrating differently from grande for a single deletion, and more than one for multiple deletions) and by comparing petite and grande mitochondrial DNA restriction maps, the petite clones could be classified into two main groups: (1) petites representing a single deletion of grande mitochondrial DNA and (2) petites containing multiple deletions of the grande mitochondrial DNA resulting in rearranged sequences. Single deletion petites may retain a large portion of the grande mitochondrial genome or may be of low kinetic cimplexity. Many petites which are scored as single continuous deletions by genetic criteria were later demonstrated to be internally deleted by restriction endonuclease analysis. Heterogeneous sequences, manifested by the presence of sub-stoichiometric amounts of some restriction fragments, may accompany the single or multiple deletions. Single deletions with heterogeneous sequences remain useful for mapping if the low concentration sequences represent a subset of the stoichiometric bands. Using a group of petites which retain single continuous regions of the grande mitochondrial DNA, we have physically mapped antibiotic resistance and mit- markers to regions of the grande restriction map as follows: C (99.3--1.4 map units)--OXI-1 (2.5--15.7)--OXI-2 (18.5--25)--P (28.1--34.2)--OXI-3 (32.2--61.2--OII (60--62)--COB (64.6--80.8--0I (80.4--85.7)--E (95--98.9).