Restriction site polymorphisms in the pig beta-globin gene cluster

A restriction fragment length polymorphism was detected in pig DNA digested with Hind III restriction endonuclease and probed with rabbit beta 1-globin gene. Eight different phenotypes were observed and for six of them family data demonstrated that they are determined by three alleles. As this polymorphism is not found with four other restriction endonucleases (Bam HI, Eco RI, Kpn I, and Pst I), single point mutations are proposed to explain the observed differences.     

Comparison of the time-mortality response of Heliothis zea to 14 isolates of Heliothis nuclear polyhedrosis virus

Twelve singly embedded isolates (SEV) and two multiply embedded isolates (MEV) of nuclear polyhedrosis viruses from Heliothis larvae were compared by time-mortality assays in neonate H. zea larvae. The isolates could be separated into six groups based on differences in the 50% survival time (ST₅₀) values. Isolates with identical restriction endonuclease (REN) profiles did not differ significantly in their ST₅₀ values, whereas isolates with several different REN cleavage sites also had significantly different ST₅₀ values. With the exception of one isolate from India, the singly embedded isolates acted faster than the multiply embedded isolates.     

The number of ribosomal RNA genes in Thermus thermophilus HB8.

We have examined the number of rRNA genes in Thermus thermophilus HB8 by hybridization of Bam HI -, Hind III - and Pst I - digests of DNA to 3'- (3 2p) 23S, 16S and 5S rRNAs according to the Southern procedure. The restriction gels gave two radioactive bands with 23S and 5S rRNA. Furthermore, band positions were indistinguishable from one another when 23S and 5S rRNAs were used as probes to Bam HI and Hind III digests, indicating that each band contains sequences corresponding to the 3'-end of 23S and 5S rRNAs. The Pst I digest also gave two radioactive bands with 23S and 5S rRNAs as probes, where one band position was identical, but the other different. The 16S rRNA did hybridize with two fragments, using a Bam HI, as well as a Bam HI - Hind III double digest. The Hind III digest gave one band using 16S rRNA as a probe. It is concluded that the Thermus thermophilus HB8 chromosome carries at least two sets of genes for 23S, 16S and 5S rRNAs.     

The use of fractionation in a polyethylene glycol-dextran two-phase system for the testing and purification of restriction endonucleases

A simple procedure was developed for testing and purification of restriction endonucleases Msp I, Pst I, Bam HI, Pvu I, Pvu II that includes biomass destruction, fractionation of cell-free extracts in the aqueous two-phase (polyethylene glycol-dextran) system and chromatography oh phosphocellulose. Optimal conditions for the fractionation of Msp I, Pst I, Bam HI, Pvu II, EcoR I, EcoR II, BspR I, Alu I were chosen. For separation of Pvu I and Pvu II gel filtration through biogel A-0.5 m was additionally introduced.     

Location of the cleavage sites on the SV 40 DNA map produced by the restriction endonucleases Pst1 and Bam1

Restriction endonucleases from Providencia stuartii (Pst 1) and Bacillus amyloliquefaciens H (Bam 1) cleave SV 40 DNA at two and one specific sites, respectively. Using EcoRI and Hind III endonuclease restriction sites as reference, the two Pst I sites were mapped at 0.050; 0.265 and the Bam I site was mapped at 0.170 of the genome length, clockwise, from the single EcoRI cleavage site.     

Characterization and Cross-transmission of Baculoviruses Infectious to the Diamondback Moth, Plutella xylostella, and some other Lepidopteran Pests of Brassica Crops

Isolates of a granulovirus (GV) from the Diamondback Moth, Plutella xylostella, and nucleopolyhedrovirus (NPV) isolates from Galleria mellonella and Autographa californica were characterized by restriction endonuclease analysis of viral DNA. The capacity for these viruses to infect P. xylostella larvae and some other lepidopteran pests of brassica crops (including Heliothis virescens, Crocidolomia binotalis and Mamestra brassicae) was examined in cross-transmission experiments in which the DNA isolated from purified progeny viruses, was compared by restriction endonuclease analysis with DNA from the inoculum viruses. Two P. xylostella GV isolates from Taiwan and China (Px GV-Taiwan and Px GV-China) appeared to be very closely-related on the basis of comparative restriction endonuclease analysis of viral genomic DNA. However, both virus isolates could be distinguished by 1-3 major band differences and by sub-molar band variation when their DNA was analysed following digestion with Eco RI, Bam HI and Hin dIII. Both P. xylostella GV isolates proved to be infectious for P. xylostella larvae but did not appear to infect M. brassicae, C. binotalis or H. virescens larvae. In contrast, a G. mellonella NPV (Gm NPV) isolate was infectious for P. xylostella larvae as well as for larvae of M. brassicae, C. binotalis and H. virescens. The results also confirmed that P. xylostella larvae are susceptible to infection by A. californica NPV. These studies form the basis for further evaluation of Px GV and Gm NPV as potential biological control agents for the Diamondback Moth.     

Restriction enzyme analysis of Listeria monocytogenes strains associated with food-borne epidemics

Listeria monocytogenes (serotype 4b) has caused four major food-borne epidemics in North America. In this study, L. monocytogenes isolates from the Nova Scotia (Canada), Boston (Mass.), and Los Angeles (Calif.) outbreaks were examined by restriction enzyme analysis with the endonuclease HhaI. Human isolates (n = 32) from the 1981 Canadian outbreak were compared with a strain recovered from coleslaw, which was epidemiologically incriminated as the vehicle of infection. After HhaI digestion, 29 of 32 isolates exhibited the restriction enzyme pattern of the reference coleslaw isolate. The restriction enzyme patterns of the nine clinical isolates from the 1983 Massachusetts outbreak were identical to each other but differed from those of raw milk isolates recovered from sources supplying the pasteurizer. Isolates (n = 48) from the 1985 California outbreak were evaluated. The restriction enzyme patterns of the L. monocytogenes isolates from humans and from the suspect cheese samples were identical to those of four of five cheese factory environmental isolates. Isolates from each of these outbreaks exhibited a restriction enzyme pattern that was characteristic of that outbreak. The case with which restriction enzyme analysis can be applied to all serotypes of L. monocytogenes argues for its use in the epidemiology of L. monocytogenes.     

Restriction enzyme analysis of Listeria monocytogenes strains associated with food-borne epidemics.

Listeria monocytogenes (serotype 4b) has caused four major food-borne epidemics in North America. In this study, L. monocytogenes isolates from the Nova Scotia (Canada), Boston (Mass.), and Los Angeles (Calif.) outbreaks were examined by restriction enzyme analysis with the endonuclease HhaI. Human isolates (n = 32) from the 1981 Canadian outbreak were compared with a strain recovered from coleslaw, which was epidemiologically incriminated as the vehicle of infection. After HhaI digestion, 29 of 32 isolates exhibited the restriction enzyme pattern of the reference coleslaw isolate. The restriction enzyme patterns of the nine clinical isolates from the 1983 Massachusetts outbreak were identical to each other but differed from those of raw milk isolates recovered from sources supplying the pasteurizer. Isolates (n = 48) from the 1985 California outbreak were evaluated. The restriction enzyme patterns of the L. monocytogenes isolates from humans and from the suspect cheese samples were identical to those of four of five cheese factory environmental isolates. Isolates from each of these outbreaks exhibited a restriction enzyme pattern that was characteristic of that outbreak. The case with which restriction enzyme analysis can be applied to all serotypes of L. monocytogenes argues for its use in the epidemiology of L. monocytogenes.     

Restriction site polymorphisms in the pig β-globin gene cluster

A restriction fragment length polymorphism was detected in pig DNA digested with Hind III restriction endo nuclease and probed with rabbit β₁-globin gene. Eight different phenotypes were observed and for six of them family data demonstrated that they are determined by three alleles. As this polymorphism is not found with four other restriction endo nucleases (Bam HI, Eco RI, Kpn I, and Pst I), single point mutations are proposed to explain the observed differences.     

A comparison of genomes of sheep pox virus isolates

Sheep pox virus DNAs from field isolates and vaccine strains were analysed by digestion with the restriction enzymes PstI and Bam HI. The restriction profiles generated by these enzymes show very close relationship between isolates from different geographical regions. The patterns of isolates can be grouped by the animal of origin (e.g. a cattle pox isolate can be differentiated from sheep pox isolate). The molecular weights of the genomes of different isolates varied from 91 to 94 MDa. The restriction enzyme patterns can be used as a molecular epidemiological tool for differentiating field and vaccine isolates.     

Comparative evaluation of plasmid profiling and ribotyping in the analysis of Lactobacillus plantarum strain heterogeneity in silage

F. DUFFNER AND M. O'CONNELL. 1995. Seventy-two Lactobacillus plantarum isolates were recovered from six uninoculated grass silages for the purposes of firstly evaluating the usefulness of (1) restriction endonuclease digestion of total genomic DNA, (2) plasmid profiling and (3) ribotyping in Lact. plantarum strain differentiation and secondly, examining the strain heterogeneity in well preserved silage.
The three methods for differentiation were applied to 72 of the isolates and allowed at least 32 different strains to be identified. Twenty-five different plasmid profiles were detected (26 if the absence of plasmids is included as a profile). Ribotyping with Eco RI identified only 11 patterns among the silage isolates. A variety of restriction enzymes was screened to increase the sensitivity of ribotyping to detect strain differences and Bam HI was used successfully for this purpose, differentiating all of the strains tested.
Two dominant strains (I and II) were identified in one particular silage, comprising 47% and 17% respectively of the isolates, while strains III and V comprised 37% and 25% of the Lact. plantarum population isolated from another of the silages.     

An alternative approach for screening active bam HI variants: overexpression in T-7 RNA polymerase based system

The type II restriction endonuclease, Bam HI, has been overexpressed in E. coli by cloning the Bam HI gene in frame with an E. coli Ribosome Binding Site (RBS) under the T7 promoter of an E. coli expression vector pRSET A. The expression level of Bam HI endonuclease using this construct was found to be higher than that reported of the overexpressing clone pAEK14. Our overexpressing clone, pAABRw in BL21 cells in presence of Bam HI methylase in pMAP6 following induction with IPTG yields about 9.2 x 10(6) units per gram wet cell paste. In vivo activity of the recombinant endonuclease could be confirmed by the SOS induction assay in JH139 cells even in the absence of T7 polymerase and cognate Bam HI methylase because of leaky expression in E. coli. This provides an alternate way to screen the active endonuclease and its variants.     

HLA-DR4-associated haplotypes are genotypically diverse within HLA

Biochemical diversity among products of class II HLA genes has been observed in individuals who appear to be HLA-D and DR-identical by cellular and serologic typing. We used techniques of restriction enzyme fragment analysis by Southern blotting to analyze this diversity at the level of cellular DNA. A panel of 17 HLA-DR4 homozygous cell lines (HCL) were investigated by using cDNA probes homologous to DQ beta, DQ alpha, and DR beta genes. Each probe was hybridized to cellular DNA digested with a series of different restriction endonucleases. Polymorphisms were observed with the use of the enzymes Pst I, Hind III, and Bam HI: Hybridization of cellular DNA digested with Hind III and Pst I with the DQ beta probe revealed specific polymorphisms, as did hybridization of the Pst I digest with the DQ alpha cDNA probe and the Bam HI digest with the DR beta probe. The observed differences fall into two categories: first, considerable diversity was seen between HLA-DR4 HCL that represent different HLA-D-defined haplotypes; second, diversity was also observed among HCL of the same DR4-associated HLA-D cluster. In contrast to the DQ cDNA probes, hybridization with the DR beta probe revealed relatively limited polymorphism by using a panel of different restriction endonucleases. Thus, although there is a general pattern of polymorphic restriction enzyme fragments homologous to DQ probes within an HLA-D cluster, the pattern seen for any particular cell line was not sufficiently distinct to assign an HLA-D or DR specificity.     

Biochemical characterization of three nucleopolyhedrovirus isolates of Spodoptera exigua and Mamestra brassicae

Three nucleopolyhedroviruses (NPVs), of different geographical origins, were biochemically characterized and compared. Two that were isolated from Spodoptera exigua (Se-UZB and Se-SP3) in Uzbekistan and Spain were SeMNPV type, and the third which was isolated from Mamestra brassicae (Mb-PL) in Poland was MbMNPV type. The Spanish isolate Se-SP3 showed restriction endonuclease (REN) profiles that were closely related to two previously described Spanish strains Se-SP1 and Se-SP2, but had some unique and characteristic REN fragments. On the other hand, comparison between the Uzbekian (Se-UZB) and the Spanish (Se-SP1, Se-SP2, and Se-SP3) isolates from S. exigua showed unrelated REN profiles. However, the Pst I and Bgl II profiles of Se-UZB and Mb-PL were identical, and very similar to the REN profiles of the MbMNPV strain, which constitutes the active component of Mamestrin^® (NPP, Nagueres, France), a commercial bioinsecticide. It is therefore very likely that the Se-UZB samples were cross-infected by the Polish strain (Mb-PL). This work presents two new strains of SeMNPV and MbMNPV, called Se-SP3 and Mb-PL, respectively, which were surveyed in two distant areas.     

A single gene encoding the fiber is responsible for variations in virulence in the fowl adenoviruses.

Intertypic recombinant fowl adenoviruses (FAVs) were generated to determine regions of the viral genome involved in virulence. Recombinants were produced with two serotype 8 FAVs, mildly virulent CFA 3 and hypervirulent CFA 40. Restriction endonuclease fragments from the genomes of the two FAVs were used to transfect primary chicken kidney cells. Virulence testing of these recombinants located the region responsible for differences in virulence to an 8.4-kb fragment of the genome located between kb 26.6 and 35.0. According to data available for a serotype 10 FAV that had been partially characterized in the laboratory, this segment of the genome contained three genes of known identity (100K, 33K, and pVIII) and a region between kb 31 and 35 with unknown coding potential (although this information subsequently became available for a serotype 1 FAV, CELO). Therefore, the region between kb 30.5 and 34.5 was sequenced. The results revealed that the unknown region encoded a fiber gene on the right strand and several small open reading frames of unknown identity on the left strand. Further recombinant viruses containing defined exchanges within the 4-kb fragment were constructed, and virulence testing of these viruses indicated that the fiber was responsible for differences in virulence for CFA 40 and CFA 3.     

Comparative restriction analysis of chromosomal DNA of strains of Photobacterium leiognathi

Chromosomal DNA in 5 hereditary variants occurring in Photobacterium leiognathi population was subjected to restriction analysis. The variants differed in the levels and regulation of luminescence and colony morphology. Agarose electrophoresis of DNA fragments isolated after exposure to Hind II, Bam HI, Bgl I and Pst I restriction endonucleases revealed respectively 38, 28, 35 and 29 fragments equally distributed by their molecular weights. Electrophoregrams of the 5 strains were absolutely identical. After exposure of DNA of all the strains to PVu II, Xho II, Sal GI and Eco RI restriction endonucleases there were detected no fragments. The pleoiotropic genetic variation in these strains was not associated with large deletions or amplification of chromosomal DNA regions.     

Estimating size and diversity of nitrifying communities in deodorizing filters using PCR and immunofluorescence

Thirty isolates of Listeria monocytogenes and 18 of L. innocua obtained from different short-ripened cheeses manufactured in Asturias (northern Spain), were compared with each other and with reference strains using serotype, phage type and pulsed-field restriction endonuclease digestion profiles analysis of the total DNA. Restriction enzymes Apa I and Sma I defined five clusters in L. monocytogenes ( m1 to m5 ) and two main clusters in L. innocua ( i1 and i2 ). Cluster i2 was further arranged into three subclusters ( i2a , i2b and i2c ) based on the different Eco 52I ( Xma III) and Crf 42I ( Sac II) patterns of its isolates. Clusters of L. innocua were clearly different whereas those of L. monocytogenes were more closely related to each other. In this latter species, serotype 4b isolates ( m4 and m5 ) constituted a more homogeneous group than serogroup 1 isolates ( m1 , m2 and m3 ). Cluster m3 contained two strains of serotype 1/2a whereas m1 and m2 harboured strains of both serotypes, 1/2a and 1/2b. Therefore, the combined use of restriction patterns and serotype may be useful to differentiate L. monocytogenes strains showing identical restriction profiles but differing in serotype. The cheese source of Listeria strains proved that isolates from cluster m1 were repeatedly detected as a contaminant in the same type of cheese. Comparison of L. monocytogenes Apa I profiles showed a genetic proximity of m4 and m5 to the recognized pathogenic strains ATCC 13932 and NCTC 11994, responsible for meningitis cases in other countries. Finally, bacteriophage typing data indicated that m4 , the sole phage typable group, had a phage type resembling that of strains causing the Auckland (New Zealand) outbreak of listeriosis in 1969. These data suggest a wide distribution of closely related types which might cause, under several circumstances, sporadic cases of listeriosis.     

SINPV基因组酶切图谱及多角体基因的序列分析

用限制性内切酶ＥｃｏＲＩ、ＸａｂＩ、ＸｈｏＩ、ＢａｍＨＩ、ＰｓｔＩ、ＳａｃＩ、ＨｉｄｎⅢ、ＳｍａＩ酶解斜纹夜蛾核多角体病毒广州株基因组ＤＮＡ,分别得到２６、２６、２４、２０、１３、１７、９、１条片段,并算得基因组平均大小为１３６．０ｋｂｐ。以ＡｃＮＰＶ多角体基因的部分读码框为探针,经Ｓｏｕｔｈｅｍ杂交将ＳＩＮＰＶ多角体基因定位于ＸｂａＩＯ片段上。将此片段克隆并序列分析,结果表明ＳＩ     

Chromosomal aberrations induced by the restriction endonucleases EcoR I, Pst I, Sal I and Bam HI in CHO cells

4 widely used cohesive end-producing restriction endonucleases (REs), EcoR I, Pst I, Sal I and Bam HI were tested in CHO cells for their aberration-inducing effects. It was demonstrated that all these REs significantly increased the frequencies of aberrant cells, the aberration frequencies per cell and the aberration frequencies per chromosome. The effects of REs on chromosomal aberrations are similar to ionizing radiation, but more minutes and interchange figures are observed. Polyploid cells are more susceptible to RE treatment, an interesting finding which may be explained by the mechanisms leading to the formation of polyploid cells.     

Purification and differentiation of three avian adenoviruses by restriction endonuclease analysis

A rapid method of ultracentrifugation pelleting of avian adenovirus (AAV) from small volume of chloroform treated infected cell culture fluid or allantoic fluid was adapted for isolation of adenoviral DNA. The viral DNA extracted from semipurified viruses was found to be intact on agarose gel and pure enough (A260/280 = 1.85-1.92) for restriction enzyme analysis. Restriction endonuclease analysis of Indian strain of AAV serotype 1, AAV serotype 4 (group I AAVs) and egg drop syndrome-76 (EDS-76) virus genomes (group III AAV) with Hind III enzyme differentiated these viruses. The AAV serotype 1 and serotype 4 strain exhibited identical Hind III profile to European viral strains belonging to same serotypes however, the EDS-76 virus gave similar but not identical profile. The calculated genomic lengths for AAV serotype 1 and EDS-76 virus were approximately found to be 33.9 and 44.4 Kb, respectively.