HindIII Digestion Research Articles

B-Cell Non-Hodgkin’s Lymphoma Cell Line (Karpas 1106) With Complex Translocation Involving 18q21.3 but Lacking BCL2 Rearrangement and Expression

Abstract A B-cell non-Hodgkin's lymphoma (B-NHL) cell line (Karpas 1106) with an unusual three-way translocation involving 18q21.3 has been derived from a patient with mediastinal lymphoblastic B-NHL. Although conventional cytogenetics showed a derivative 18q-identical to that seen in cases with t(14;18)(q32.3;q21.3), no translocations of either chromosome 14 could be detected. Instead fluorescent in situ hybridization analysis using a chromosome-18 paint showed that the segment 18q21.3–18qter had become sandwiched on a derivative chromosome X between segments Xqter-c- Xq28 and 13q12-qter, with the centrometric site of 18q21.3 subband juxtaposed to the X sequences. Pulsed-field DNA blots failed to detect rearrangement of the BCL2 gene. Conventional DNA blots using a variety of restriction digests and both 52 and 32 BCL2 and FVT 1 probes also failed to detect rearrangement in Karpas 1106. A rearranged fragment seen only in HindIII digests with 52 BLC2 probes may represent a local microalteration, which is either a mutation or small deletion involving the HindIII site as seen in other cases of B-NHL. Neither BCL2 RNA nor BCL2 protein expression were detected. These and other data suggest that genes at 18q21.3, other than BCL2 and FVT1, may be targets for translocation in certain subgroups of B-NHL.

Chloroplast DNA differences between cultivated hop, Humulus lupulus and the related species H. japonicus

Chloroplast DNA (cpDNA) of Humulus Lupulus and H. japonicus was examined by restriction endonuclease analysis with BamHI, BanI, BclI, BstEII, DraI, EcoRI, EcoRV, HindIII, KpnI, PaeR7I, PstI, PvuII, SalI and XhoI. The restriction fragment patterns showed that the cpDNAs shared a large number of restriction sites. However, the chloroplast genomes of the two species could be distinguished by differences in restriction site and restriction fragment patterns in the PstI, PvuII, BclI, EcoRV, DraI and HindIII digests. On the basis of the complexity of restriction enzyme patterns, the enzymes PstI, PvuII, SalI, KpnI and XhoI were selected for mapping the chloroplast genomes. Single and double restriction enzyme digests of cpDNA from the two species were hybridized to cpDNA probes of barley and tobacco. The data obtained from molecular hybridization experiments were used to construct the cleavage site maps. Except for the PstI digest, the arrangement of cpDNA restriction sites was found to be the same for both species. An extra PstI site was present in H. lupulus. Three small insertions/deletions of about 0.8 kbp each were detected in the chloroplast genomes of the two species. Two of these insertions/deletions were present in the large and one in the small singlecopy region of the chloroplast genome. The cpDNA of Humulus was found to be a circular molecule of approximately 148 kbp that contains two inverted repeat regions of 23 kbp each, a small and a large single -copy region of approximately 20 kbp and 81 kbp, respectively. The chloroplast genome of hop has the same physical and structural organization as that found in most angiosperms.

Dysfunctional growth hormone receptor in a strain of sex-linked dwarf chicken: evidence for a mutation in the intracellular domain

The sex-linked dwarf (dwdw) chicken represents a valuable animal model for studying GH insensitivity and the consequence of mutations in the GH receptor (GHR) gene. We have recently reported undetectable hepatic GH-binding activity and an aberrantly sized transcript in a strain of dwdw chickens obtained from Arbor Acre Farms, Inc. (Glastonbury, CT, USA). Southern blot analysis of the chicken GHR (cGHR) gene revealed a restriction-fragment length polymorphism in HindIII and EcoRI digests of genomic DNA in this strain of dwdw chicken. In order to localize the molecular mutation, we analysed the gene structure and determined the complete sequence of the 3' untranslated region (3' UTR) of the normal cGHR. With the use of this information, we located a large deletion in the 3' end of the cGHR gene of the Connecticut (CT) strain of dwdw chicken. This deletion (1773 bp) contained 27 highly conserved amino acids of the 3' end of the coding region, the in-frame stop codon, a less frequently used poly(A) signal that is normally found 445 bp downstream of the stop codon, and a large portion of the 3' UTR. Because of this deletion, 27 novel amino acids were substituted and the open reading frame was extended for an additional 26 amino acids before reaching the transcriptional termination site. The predicted amino acid sequence of the novel carboxyl-terminus of the dwdw cGHR is largely hydrophobic with a polylysine tail, whereas the carboxyl-terminus of the wild-type (DwDw) cGHR is composed of hydrophilic amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of restriction endonuclease analysis, ribotyping, and pulsed-field gel electrophoresis for molecular differentiation of Clostridium difficile strains

A combined clinical and molecular epidemiologic analysis of 46 strains of Clostridium difficile, including 16 nosocomial isolates from one ward (outbreak ward) plus 17 other nosocomial isolates and 13 community-acquired isolates, was performed. HindIII digests of total cellular DNA were analyzed by restriction enzyme analysis (REA) and ribotyping; SmaI digests were analyzed by pulsed-field gel electrophoresis (PFGE). Isolates were assigned to typing groups on the basis of the profiles detected; isolates with closely related profiles were assigned to subgroups. The 16 isolates from the outbreak ward were resolved by both REA and PFGE into five distinct groups; 13 isolates represented two REA groups and three PFGE groups and two isolates were resolved as distinct groups by both techniques. DNA obtained from one isolate was persistently partially degraded, precluding analysis by PFGE. Seventeen sporadic nosocomial isolates were resolved by REA and PFGE into comparable numbers of groups (i.e., nine groups) and subgroups (i.e., 15 and 14 subgroups, respectively), with two isolates not evaluable by PFGE. The 13 epidemiologically unrelated community-acquired isolates were assigned to 11 groups by REA and to 12 groups by PFGE. Overall, ribotyping identified only nine groups among the 46 isolates. We conclude that REA and PFGE have comparable discriminatory powers for epidemiologic typing of C. difficile isolates and that ribotyping is appreciably less discriminatory. For a few isolates, partial DNA degradation prevented analysis by PFGE but not by REA or ribotyping; the cause of the degradation is unknown.

Expression of transcobalamin II mRNA in human tissues and cultured fibroblasts from normal and transcobalamin II-deficient patients.

Transcobalamin II (TCII) is an important plasma transporter of cobalamin (Cbl; vitamin B12). In the present study, TCII gene expression in human and rat tissues and in the fibroblasts of patients with TCII deficiency was investigated. Northern-blot analyses revealed expression of TCII mRNA in many human and rat tissues. In humans, this was 14-fold higher in the kidney than in liver, whereas in the rat the levels of expression were similar in the kidney and liver. Southern-blot analysis of genomic DNA from several species revealed sequence similarity in TCII across species. Metabolic labelling and ribonuclease protection assay revealed a 43 kDa TCII protein and a fully protected TCII mRNA band in normal fibroblasts but not in fibroblasts from three TCII-deficient patients. Southern-blot analysis of genomic DNA from all these fibroblasts revealed identical restriction patterns on BamHI, HindIII, KpnI, MspI and EcoRI digestion. On the basis of these results, we suggest that TCII is expressed in multiple tissues, and its level of expression in tissues varies within the same and across species. Furthermore, the TCII deficiency characterized in this study is due to the absence of TCII protein which in turn is due to the absence or extremely low levels of its mRNA and not to detectable gross alterations in the gene structure.

Characterization of the DNA of a Nuclear Polyhedrosis Virus Selected for an Increased Rate of Vertical Transmission

A strain of Spodoptera frugiperda nuclear polyhedrosis virus (SfNPV) was selected in previous research for an increased rate of vertical transmission by isolating it from pupae that had been infected through vertical transmission. Differences were detected between the wild (PW) and the selected (PS) isolates of SfNPV when the viral DNA was digested with the restriction enzymes EcoRI, HindIII, and BamHI. The restriction profiles indicated a possible DNA insertion in the PS isolate. Probes with 32 P-labeled SfNPV DNA and S. frugiperda cellular DNA indicated that there was only viral DNA, not S. frugiperda cellular DNA, in the genome of the PS isolate. Therefore, the differences between the PW and the PS isolates were due to rearrangement of DNA within the viral genome. Cluster analysis based on numbers of DNA fragments shared among SfNPV isolates from the EcoRI, HindIII, and BamHI enzyme digests indicated that the PS isolate had one of the more unique genomes when compared with the PW isolate and 22 other SfNPV isolates analyzed in previous research. However, the PS DNA enzyme digests did not fall outside the range of patterns of digests observed in the natural isolates.

DNA Sequencing Directly from a Mixture Using Terminal-Base-Selective Primers

DNA cloning is often used to select and amplify one DNA species from a mixture. However, the cloning process is complex and labor-intensive. We have developed a new two-step method for DNA sequencing directly from a mixture. The first is the introduction of a known oligonucleotide (common part) into the terminus of unknown DNA by ligation. The second is selective DNA sequencing using primers with two additional nucleotides at the 3' terminus in addition to the common part (terminal-base-selective primers). The primers work only for templates on which the primers perfectly hybridized. This method was found to be effective for the HindIII digestion products of lambda phage.

A molecular and cytogenetic analysis of λ 20p7 fragment DNA from the proximal β-heterochromatin of Drosophila melanogaster

A DNA fragment from the Drosophila melanogaster genome, cloned in /g120p7, was derived independently from clones /g120 and λL [Baiborodin et al., Genetika 29 (1993) 403–416; Sharakhov et al., Genetika 29 (1993) 392–402]. In situ hybridization of λ.20p7 DNA to the chromosomes of D. melanogaster demonstrated preferential hybridization of the fragment to the chromocenter of polytene chromosomes and to pericentric heterochromatin of chromosomes II, IV and X at the metaphase plate. Copy number per haploid genome for /g120p7 was estimated as approximately 200. Based on Southern blotting, the major portion of this moderate repeat was localized in the region of a 5.5-kb HindIII digest. In situ hybridization to polytene chromosomes from strain fs(2)β trophocytes revealed that repeats homologous to A,20p7 are located in the proximal heterochromatin which undergoes structural reorganization during tissue differentiation. The nucleotide sequence of two segments of the clone /g120p7, Dm0.9 and Dm270, was determined. Sequence analysis of the 300-bp Dm0.9 clone revealed that it contains 21-bp and 30-bp d(GT/CA) sequences, a 12-bp AT box, recognition sites for nuclear factors NFI and Spl, and a set of inverted repeats. Clone Dm270 contains an open reading frame (ORF). The deduced amino acid (aa) sequence shares homology with the gag-like gene from type-I (Rl) ribosomal DNA insertion and may code for a polypeptide of 10 kDa. The Dm270 sequence was found to contain two direct repeats showing homology to the human CENP-B box.

Co-elimination of mec and spa genes in Staphylococcus aureus and the effect of agr and protein A production on bacterial adherence to cell monolayers.

Phenotypic loss of protein A production was tested in six methicillin-resistant (McR) Staphylococcus aureus (MRSA) isolates and their isogenic methicillin-sensitive (McS) variants by a radiolabelled IgG-binding assay with washed cells and by Western blotting of supernates prepared from lysed washed cells. Genomic DNA was probed for homology with the protein A gene (spa) in EcoRI digests and for homology to the methicillin resistance gene (mec) in HindIII digests. The McS variants had lost homology with mec. An isogenic pair of McR and McS strains, and derivatives of S. aureus 8325-4 with site-specific mutations of the accessory gene regulator locus (agr) and spa, were tested for adherence to human peritoneal mesothelial cells in monolayer culture. The isogenic pair were also tested for adherence to HEp-2 and Vero cell monolayers in assays with 3H thymidine-labelled bacteria. McR isolates produced protein A which was absent from three strains that had become McS. This correlated with deletion of the spa locus. Spa homology, but reduced production of protein A, was retained in one McS strain which also showed reduced adherence to HEp-2, Vero and mesothelial cells (p < 0.05) compared with the parent McR strain. A spa mutation in strain 8325-4 did not significantly affect adherence to mesothelial cells but mutation in agr increased adherence significantly in both Spa+ and Spa- strains.

Genetic structures of the B2 and B1 Escherichia coli strains responsible for extra-intestinal infections

Escherichia coli strains causing human extra-intestinal infections may be divided into two groups, B1 and B2 according to the electrophoretic patterns of carboxylesterase B. This study compares the restriction fragment length polymorphism (RFLP) of ribosomal DNA (rDNA) for 45 B1 strains and 45 B2 strains to examine the genetic structure of B2 strains and to distinguish them from B1 strains. The isolates were chosen for diversity in their allozymes of esterases, B, A, C and I, their production of virulence factors (alpha-haemolysin, mannose resistant haemagglutinin and cytotoxic necrotizing factor) and certain O antigens, and their pathological and geographical origins. DNA was digested with HindIII and BamHI restriction enzymes and analysed by Southern blotting. The resulting rDNA RFLP patterns of B2 strains were distinct from those of the B1 strains. Moreover, the B2 strains appeared to be less heterogeneous than the B1 strains. The B2 strains gave 13 ribotypes (resulting from the combination of the rDNA RFLP patterns obtained with HindIII and BamHI digestions) while the B1 strains gave 32 ribotypes. Correspondence analysis of the data showed that several clusters of strains were identified in the B2 strains by particular ribotypes, certain associations of esterase B and A electrophoretic variants, O serotypes and virulence factor production. In contrast, these parameters appeared to be unrelated in the B1 strains, reflecting their heterogeneity. These findings, which differentiate two levels of genetic heterogeneity within E. coli pathogenic isolates, indicate that the B2 strains constitute a phylogenetically distinct group within the species.

Characterization of five Indian isolates of the nuclear polyhedrosis virus ofHelicoverpa armigera

Five Indian geographical isolates of the nuclear polyhedrosis virus ofHelicoverpa (Heliothis) armigera were studied for the variation in their size, virulence, and the restriction and electrophoretic patterns of the polyhedrin. The diameter of the polyhedra varied from 1.6 to 2.4 μm (mean 1.933 μm), with no significant differences among the five isolates. The most virulent isolate was that from Ooty (OOT), with the lowest LC50 value of 2.538 × 103 purified occlusion bodies (POB)/ml, followed by the isolate from Coimbatore (CBE). Genomic variability as determined by the HindIII digestion pattern of the DNA, showed that isolates OOT and CBE were similar and that the three other isolates,viz., APK, GUJ, and RAJ represented another group which had a similar restriction pattern. The SDS-PAGE electrophoretic pattern of the polyhedrin of all the isolates was the same.

Prevalence of nptII and Tn5 in kanamycin-resistant bacteria from different environments

Kanamycin (Km)-resistant bacterial populations in different soil, river water, sewage and pig manure slurry samples were enumerated and their prevalence in the total populations determined. About 350 Km-resistant Gram-negative colonies grown in the presence of kanamycin were identified using a rapid presumptive identification scheme. They were then screened for the presence of Tn5 and nptII sequences using hybridization of cells in dot blots, of Southern-blotted genomic DNA extracts and of PCR amplification products. Colonies reacting positively with a 2.7 kb probe of the central region of Tn5, or with a 925 bp nptII specific probe were primarily obtained from sewage samples, whereas fewer were obtained from pig manure slurry, river water and soil. However, in soil samples bacteria containing Tn5 or nptII were not found. Transposon Tn5 carrying the nptII gene could be unequivocally demonstrated in 3 isolates from sewage, identified as Aeromonas spp. (2x) and Escherichia coli. HindIII digests of chromosomal DNA obtained from these strains were cloned and shown to confer Km resistance to a sensitive E. coli strain. Further, various strains revealed the presence of nptII homologous sequences in a non-Tn5 background. The occurence of Tn5 and nptII in the samples was also assessed via PCR analysis of total community DNA extracts obtained from the aforementioned environmental samples. Evidence for the occurence of nptII was obtained for sewage, pig manure slurry, for 2 (out of 3) river water (Avon, Rhine) and 3 (out of 6) soil (Flevo silt loam, Westmaas silt loam, Ahlum rhizosphere) samples. Tn5 was not detectable via PCR in any of these environmental DNA extracts but it was found in Ede loamy sand and Flevo silt loam samples taken from a field microplot 2 and 4 weeks after release of a Tn5-containing genetically modified organism.

Prevalence of nptII and Tn5 in kanamycin-resistant bacteria from different environments

Kanamycin (Km)-resistant bacterial populations in different soil, river water, sewage and pig manure slurry samples were enumerated and their prevalence in the total populations determined. About 350 Km-resistant Gram-negative colonies grown in the presence of kanamycin were identified using a rapid presumptive identification scheme. They were then screened for the presence of Tn5 and nptII sequences using hybridization of cells in dot blots, of Southern-blotted genomic DNA extracts and of PCR amplification products. Colonies reacting positively with a 2.7 kb probe of the central region of Tn5, or with a 925 bp nptII specific probe were primarily obtained from sewage samples, whereas fewer were obtained from pig manure slurry, river water and soil. However, in soil samples bacteria containing Tn5 or nptII were not found. Transposon Tn5 carrying the nptII gene could be unequivocally demonstrated in 3 isolates from sewage, identified as Aeromonas spp. (2x) and Escherichia coli. HindIII digests of chromosomal DNA obtained from these strains were cloned and shown to confer Km resistance to a sensitive E. coli strain. Further, various strains revealed the presence of nptII homologous sequences in a non-Tn5 background. The occurence of Tn5 and nptII in the samples was also assessed via PCR analysis of total community DNA extracts obtained from the aforementioned environmental samples. Evidence for the occurence of nptII was obtained for sewage, pig manure slurry, for 2 (out of 3) river water (Avon, Rhine) and 3 (out of 6) soil (Flevo silt loam, Westmaas silt loam, Ahlum rhizosphere) samples. Tn5 was not detectable via PCR in any of these environmental DNA extracts but it was found in Ede loamy sand and Flevo silt loam samples taken from a field microplot 2 and 4 weeks after release of a Tn5-containing genetically modified organism.

Comparison of allozyme, RFLP, and RAPD markers for revealing genetic variation within and between trembling aspen and bigtooth aspen

We examined genetic variation in allozyme loci, nuclear DNA restriction fragment length polymorphisms (RFLPs), and random amplified polymorphic DNAs (RAPDs) in 130 trembling aspen (Populus tremuloides) and 105 bigtooth aspen (P. grandidentata) trees. In trembling aspen 10 out of 13 allozyme loci assayed (77%) were polymorphic (P), with 2.8 alleles per locus (A) and an expected heterozygosity (He) of 0.25. In contrast, bigtooth aspen had a much lower allozyme genetic variability (P=29%; A=1.4; He=0.08). The two species could be distinguished by mutually exclusive alleles at Idh-1, and bigtooth aspen has what appears to be a duplicate 6PG locus not present in trembling aspen. We used 138 random aspen genomic probes to reveal RFLPs in HindIII digests of aspen DNA. The majority of the probes were from sequences of low copy number. RFLP results were consistent with those of the allozyme analyses, with trembling aspen displaying higher genetic variation than bigtooth aspen (P=71%, A=2.7, and He=0.25 for trembling aspen; P=65%, A=1.8, and He=0.13 for bigtooth aspen). The two species could be distinguished by RFLPs revealed by 21 probes (15% of total probes assayed). RAPD patterns in both species were studied using four arbitrary decamer primers that revealed a total of 61 different amplified DNA fragments in trembling aspen and 56 in bigtooth aspen. Assuming a Hardy-Weinberg equilibrium, estimates of P=100%, A=2, and He=0.30 in trembling aspen and P=88%, A=1.9, and He=0.31 in bigtooth aspen were obtained from the RAPD data. Five amplified DNA fragments were species diagnostic. All individuals within both species, except for 2 that likely belong to the same clone, could be distinguished by comparing their RAPD patterns. These results indicate that (1) RFLPs and allozymes reveal comparable patterns of genetic variation in the two species, (2) trembling aspen is more genetically variable than bigtooth aspen at both the allozyme and DNA levels, (3) one can generate more polymorphic and species-specific loci with DNA markers than with allozymes in aspen, and (4) RAPDs provide a very powerful tool for "fingerprinting" aspen individuals.

Molecular analysis in patients with mucopolysaccharidosis type II suggests that DXS466 maps within the Hunter gene.

Hunter disease is an X-linked mucopolysaccharidosis caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). Using the IDS cDNA and DNA probes corresponding to loci flanking the IDS locus, we performed molecular genetic studies in two patients with Hunter syndrome. An interstitial deletion spanning the middle part of the IDS gene was found in the first patient. The second patient carries a gross gene rearrangement that can be detected after HindIII or EcoRI digestion of genomic DNA, and is similar to that found recently in seven unrelated Hunter patients. Our data suggest that the structural aberration observed is a partial intragenic inversion. As the same altered hybridization pattern is also revealed by the recently described anonymous DNA probe II-10, which recognizes a frequent TaqI restriction fragment length polymorphism at the DXS466 locus, we conclude that DXS466 maps within the IDS gene, probably in an intron.

Nucleotide sequence of a soybean chalcone synthase gene with a possible role in ultraviolet-B sensitivity, Gmchs6.

Recent trends in stratospheric ozone depletion and projected increases in solar UV-B radiation (280-320 nm) have intensified studies of the ecological and physiological effects of increased levels of UV-B on higher plants (Caldwell, 1981; Worrest and Caldwell, 1986). Soybean (Glycine max L. Merr) is among the most extensively studied plants because it is a key crop of worldwide importance and because its potential susceptibility to increased levels of solar UV-B has been amply documented (Teramura et al., 1990, and refs. therein). From such studies, a pair of cultivars of contrasting sensitivity to UV-B has been identified. Williams is tolerant to supplemental UV-B fluences simulating a 25% ozone depletion, whereas Essex is sensitive to the same fluences, resulting in reduction of seed yield by 20 to 25%. The possibility that this may be due to differences in UV-B-absorbing compounds has also been noted. Flavonoids are among the best candidates for UV-B-absorbing compounds, and it is well-known that they can function as UV-B protectants because of their strong molecular absorption of UV light and accumulation in the epidermal layer of leaf tissues (Caldwell et al., 1983). It is also well documented that CHS, a key enzyme of flavonoid biosynthesis, plays an important role in the accumulation of flavonoids in leaf epidermis (Cosio et al., 1985; Beerhues et al., 1988). Thus, we reasoned that the molecular basis for the contrasting sensitivity to UV-B between Williams and Essex might include differences in some aspect of a CHS gene. We have already identified seven members of the soybean chs gene family. Although it is not yet known which member of the gene family is responsible for flavonoid accumulation in leaf epidermis, one interesting discovery was made when we compared Southern hybridization patterns of Williams and Essex genomic DNA HindIII digests with CHS gene 1 probe, i.e. the intensity of the 2.0-kb HindIII band, designated as gene 6 (Gmchs6), was 4 times weaker in Essex than Williams. This suggested the possibility that gene dosage differences in gene 6 might account for the difference in UVB tolerance. The possibility that differences in intensity reflected a restriction site polymorphism did not seem very likely because of the apparent absence of an additional band corresponding to this gene on the Southern blot. However, I _ GCTAZ __ r% Z , __ GC_ 'rCC At, ' -'ITC'rTCCAT AAAGG GAAAr__ _ 'rA'_FTA --,_ , _ 7AA TTTAAa _ sf C_ _ TTG_ _TGG_ _ AC_ ATTe_ _AT C _ A-TT_A_TAAT .AT_A_,CAAI 'A,T

Categorization of clinical isolates of Helicobacter pylori on the basis of restriction digest analyses of polymerase chain reaction-amplified ureC genes.

Restriction endonuclease analyses of a 1.1-kb polymerase chain reaction-amplified portion of the ureC gene from Helicobacter pylori were used to group or to differentiate 21 clinical isolates. Isolates were placed into 4 groups after HindIII digestion alone, and placement was expanded into 15 groups after isolates were digested with AluI and PvuI.

T(8;14)(q11;q32) in acute lymphoid leukemia: Description of two cases

An 8;14 chromosome translocation with breakpoints at q11 and q32, respectively, is described as the sole abnormality in bone marrow cells of two adult patients with common acute lymphoblastic leukemia (ALL). The Southern blot analysis revealed a rearrangement after BamHI and HindIII digestion and hybridization with a JH probe, thus demonstrating the involvement of the gene coding for the heavy chains of the immunoglobulins (IgH). Therefore, a pathogenetic mechanism similar to that observed in Burkitt's lymphoma and its variants, or in other lymphomas with t(11;14) or t(14;18), may be hypothesized. In all these cases IgH is juxtaposed to an oncogene (c-MYC, BCL-1, and BCL-2, respectively). A similar structure, with oncogene type potential, could be present on 8q11. The patients underwent a complete remission after induction therapy.

Type I interferon genes in cattle: restriction fragment length polymorphisms, gene numbers and physical organization on bovine chromosome 8.

Multiple, superimposed Type I interferon (IFN) restriction fragments were resolved following 72-92 h of horizontal electrophoresis. Restriction fragment length polymorphisms (RFLPs) for alpha IFN (IFNA), beta IFN (IFNB), omega IFN (IFNW) and trophoblast IFN (IFNT) genes were identified in HindIII, EcoRI and TaqI digestions from 313 cattle. RFLPs with codominant segregation in cattle pedigrees were considered alleles, and 19 distinct polymorphic Type I IFN loci (5 IFNA, 4 IFNB, 8 IFNW and 2 IFNT) were identified. Allele frequencies and observed heterozygosity values were calculated for each locus and several loci were considered highly informative for linkage analysis. Bovine IFN gene numbers (10 IFNA, 6 IFNB, 20 IFNW and 6 IFNT) were estimated from the number of polymorphic loci plus additional monomorphic hybridizing bands present in EcoRI and HindIII digestions. Physical linkage of the Type I IFN gene families on bovine chromosome 8 was demonstrated by pulsed field gel electrophoresis (PFGE). Hybridization of two or more IFN probes to similarly sized PFGE fragments suggested the tentative gene family order: IFNA/IFNW-IFNT-IFNB. These studies provide a basis for the development of more detailed genetic and physical maps of the bovine Type I IFNs.

Ribotype analysis of Pseudomonas pseudomallei isolates.

No epidemiological typing system to differentiate among Pseudomonas pseudomallei isolates has been available. Ribotype analysis was developed and used to examine 74 clinical and 10 environmental isolates of P. pseudomallei from Thailand. Six P. pseudomallei ribotypes were identified from restriction fragment polymorphisms of EcoRI chromosomal digests. The predominant ribotype, A, was found in 59 of the isolates examined. By using patterns from hybridizations with SalI, HindIII, and PstI restriction digests, isolates of ribotype A were subdivided into a further five subtypes, giving a total of 10 differentiable P. pseudomallei types. In 23 of 34 melioidosis patients studied, multiple P. pseudomallei isolates were present. In all but one of these patients, a single ribotype of the organism was present. Isolation of two different ribotypes of P. pseudomallei from one patient, one each in sputum and urine, suggests that superinfection may have occurred. The ribotype was shown to be conserved during the course of antibiotic treatments in seven patients studied, although the antibiotic sensitivity patterns in the isolates from these patients varied. The prevalence of subtype A1 in clinical and environmental specimens suggests that this strain may be predominant in this geographical location. These results demonstrate the usefulness of the ribotyping method for epidemiological studies of P. pseudomallei.