Probe Southern Blots Research Articles

Development of a PCR assay to detect Fusarium poae in wheat

Random amplified polymorphic DNA assays were carried out on a range of isolates of F. poae to identify markers common to all isolates. Two fragments were isolated, cloned and used to probe Southern blots of DNA from F. poae and isolates from a range of wheat seed and stem base pathogens. One fragment, which hybridized preferentially to DNA of F. poae was partially sequenced and two pairs of primers (Fp8 F/R and Fp82 F/R) were generated for use in the polymerase chain reaction (PCR). Amplification of target DNA occurred following PCR of all isolates of F. poae but not from any of a range of other fungal species associated with diseases of cereal ears and seed. The primer pair Fp82 F/R was used to detect F. poae in extracts from wheat seed samples contaminated with Fusarium species. This system offers the potential to determine the presence of F. poae in wheat and avoid problems commonly associated with conventional diagnosis of the disease and isolation of the pathogen.

Molecular analysis of T-cell receptor repertoire in bone marrow transplant recipients: evidence for oligoclonal T-cell expansion in graft-versus-host disease lesions

We have analyzed the T-cell receptor (TCR) V beta repertoire using polymerase chain reaction (PCR) in a cohort of eight patients receiving allogeneic bone marrow transplantation (BMT) from related and unrelated donors at the City of Hope. Results of PCR studies from graft-versus-host disease (GVHD) skin lesions show a bias in the usage of TCR V beta families, whereas examination of peripheral blood (PB) withdrawn at the same time did not reveal a similar phenomenon. In one such family, TCR V beta 2 is predominantly expressed in 7 of 7 biopsy specimens examined. V beta 2 TCR expression from these patients was analyzed more extensively using a combination of individual TCR gene cloning, followed by sequence analysis. We found evidence of oligoclonal expansion of single V beta 2-bearing TCRs in GVHD lesions, and in the PB of some patients after diagnosis of GVHD. In contrast, GVHD-negative biopsy samples showed no evidence for clonotypic TCR amplification. Sequence-specific TCR CDR3 region probes were derived from analysis of the predominant expressed TCR in GVHD lesions, and used to probe Southern blots of amplified V beta 2 TCR mRNA from PB and tissue from BMT recipients and their respective donors. In most cases the probes are highly specific in detecting TCR expression from GVHD lesions alone, although in several instances expression could be detected in PB after GVHD diagnosis. These data provide supporting evidence for the hypothesis that acute GVHD is associated with expansion of T-cell clones expressing antigen-specific TCRs that may contribute to the disease pathology.

IDENTIFICATION OF A MYOSIN‐LIKE PROTEIN IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

ABSTRACT A myosin‐like protein was identified in vegetative cells of the unicellular green alga Chlamydomonas reinhardtii Dangeard. Polyclonal antibodies affinity purified against the heavy chain of slime‐mold myosin recognized a 180,000 Mr protein in western blots of total protein extracts from three different strains, including cyt‐1, a cytokinesis‐defective mutant. Immunoblots of isolated chloroplasts indicated that some of the cellular myosin fractionated with chloroplasts, whereas tubulin did not. Evidence for the presence of at least one myosin gene was obtained by probing Southern blots of genomic DNA with a myosin heavy‐chain gene fragment isolated from the green alga Ernodesmis verticillata (Kützing) Børgesen. Collectively, the immunological and molecular data identify at least one myosin heavy‐chain gene and a myosin‐like protein in vegetative cells of the model organism Chlamydomonas.

Hybridization and detection of digoxigenin probes on RNA blots.

AbstractThe probing of RNA gel blots (also called Northern blots) with labeled nucleic acids provides data on the relative levels of steady state gene expression, and on RNA processing. The principle of the technique was first described by Alwine et al. (1). The protocol described in this chapter demonstrates the use of nonradioactive digoxigenin-labeled probes (chapter 10 and chapter 11) on RNA blots (chapter 7 and chapter 8). The detection system employs an antidigoxigenin antibody/alkaline phosphatase conjugate, and the chemiluminescent substrate 3-(2′-spiroadamantane)-4-methoxy-4-(3″-phosphoryloxy)-phenyl-1,2-dioxetane (AMPPD) (2,3). The procedure used is similar to that described for probing Southern blots and detection by chemiluminescence in chapter 17. However, this procedure uses a different membrane for support of the immobilized nucleic acids. This, and the fact that RNA is being probed, require modifications to the hybridization buffer and washing procedure.KeywordsSodium Dodecyl SulfateHybridization BufferTrisodium CitrateSterile ContainerPrehybridization SolutionThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Major Histocompatibility Complex Class IV Restriction Fragment Length Polymorphism Markers in Replicated Meat-Type Chicken Lines Divergently Selected for High or Low Early Immune Response

Information on MHC may improve the efficiency of selection for immunological traits via the application of marker assisted selection or by selecting directly for a specific restriction fragment length polymorphism (RFLP) band or MHC haplotype. An experimental procedure is presented here for identifying MHC genes that are related to early immune response. A Class IV cDNA clone was used to probe Southern blots of erythrocyte genomic DNA from chickens. Chickens were taken from the second (S2) and third (S3) generations of replicated lines divergently selected for high antibody response (HC1, HC2) or low antibody response (LC1, LC2) to Escherichia coli vaccination at 10 days of age. These selection criteria have been found to be associated with other immunological parameters. The hypothesis that these selected lines differ in their MHC loci was evaluated by comparing the frequencies of MHC RFLP markers (single RFLP bands) and haplotypes (patterns of RFLP bands). The significant differences between LC and HC in the frequency of many MHC RFLP bands and of five MHC haplotypes indicate that early antibody production is influenced by MHC genes. The reliability of the association between the selection and frequency differences was tested and proven in most cases by analysis of the replicated lines. These differences in RFLP markers represent a change in allelic frequencies in MHC genes, probably due to selection. The results imply a connection between the Class IV genes and early antibody production, and they show the potential of prospective breeding not only by immunological phenotype but also by genotype (i.e., using RFLP markers of the MHC).

Characterization and partial nucleotide sequence of the DNA fingerprinting probe Ca3 of Candida albicans

The moderately repetitive Ca3 fragment of Candida albicans has been used as an effective DNA fingerprinting probe in epidemiological studies. EcoRI digestion of Ca3 DNA results in seven fragments of 4.2 kb (A), 2.98 kb (B), 2.85 kb (C), 0.77 kb (D1), 0.77 kb (D2), 0.38 kb (E), and 0.30 kb (F). Five of these EcoRI fragments have been mapped in the 5'-3' order C B D1 A D2. The intact Ca3 probe and the three largest EcoRI fragments, A, B, and C, were individually used to probe Southern blots of EcoRI-digested DNA of a set of test strains, transverse alternating field electrophoresis-separated chromosomes of strain 3153A, and Northern (RNA) blots of test strain 3153A. Fragments, A, B, and C each generate a different Southern blot hybridization pattern with EcoRI-digested whole-cell DNA; Ca3 sequences are present in at least five of seven separable chromosomes and a minichromosome of strain 3153A; fragments A, B, and C are distributed differently on chromosomes; and fragments A, B, and C do not cross-hybridize. Ca3 hybridizes to three major transcripts of 2.8, 2.3, and 1.5 kb. Fragment A hybridizes intensely to the 1.5-kb transcript, while fragments B and C both hybridize intensely to the 2.8- and 2.3-kb transcripts. The B fragment, which contains 2,980 bp and contributes to the major portion of the Ca3 pattern, was sequenced. Both direct and inverted repeat sequence motifs were identified. These results provide us with initial insights into the evolution of the Ca3 pattern and the nature of the probe.

Localization of the mouse Na+/H+ exchanger gene on distal chromosome 4.

A cDNA probe of the human Na+/H+ exchanger (formerly, antiporter) was used to probe Southern blots of DNA obtained from various inbred mouse strains. On the basis of fragment length polymorphisms generated by each of two restriction enzymes, three different alleles could be identified. The distribution of alleles of the Nhe-1 gene was determined in the BXD recombinant inbred strain series. Comparison with previously reported strain distribution patterns shows that the gene encoding the mouse Na+/H+ exchanger maps to distal mouse Chromosome 4, between Lck and Akp-2.

Characterization of Overexpressed cDNAs Isolated from a Hormone-Autonomous, Radiation-Induced Tumor Tissue Line of Arabidopsis thaliana

To investigate the molecular mechanisms of hormonal control of growth, we constructed a subtracted cDNA library enriched for sequences expressed more in a hormone-autonomous, radiation-induced tumor tissue line of Arabidopsis thaliana than in normal, hormone-dependent callus. Ten cDNA clones, which are expressed 1.3- to 10-fold more in the tumor line, were isolated and partially characterized. The clones differ greatly in their level of expression in tumor tissue and in their pattern of expression in plant organs. Southern blot hybridization and sequence analysis showed that this group contains three pairs of closely related clones. Northern blot analysis indicates that one pair of clones represents two members of a gene family that are expressed in different plant organs. One of the isolated sequences shows strong sequence similarity to a cDNA encoding a lipid transfer protein. Two sequences are highly similar to those of previously described membrane channel proteins but have different organ specificities. Two other cDNAs have significant sequence similarity to glycine-rich proteins and hydroxy-proline-rich glycoproteins. When used to probe Southern blots, none of the cDNAs identified polymorphisms between tumor and callus DNA, which might be expected if their overexpression were due to local genome rearrangements induced by radiation. The diversity observed among these 10 clones suggests that some are likely to be involved in tumorous growth and not simply specific to a certain cell or tissue type present in the tumor.

Micronuclear DNA from Paramecium tetraurelia: serotype 51 A gene has internally eliminated sequences.

A method for the isolation of micronuclear DNA from Paramecium tetraurelia has been developed. After cell lysis, a low speed centrifugation at 1,000 g is used to remove all of the unbroken cells and macronuclei and approximately two thirds of the macronuclear fragments. Next a higher speed centrifugation of 9,000 g sediments the micronuclei and frees them from small particulates and soluble constituents. Advantage is then taken of the fact that micronuclei have a lower density than do macronuclear fragments in 45%-60% Percoll. Micronuclei float to the top during centrifugation at 24,000 g, while macronuclear fragments sediment. After several cycles of centrifugation in Percoll, the micronuclei, although heavily contaminated with cytoplasmic components, are essentially free of macronuclei and macronuclear fragments. Micronuclear DNA can then be extracted from the suspension. The whole procedure is very rapid and in about an hour micronuclear and macronuclear DNA can be separated. About 2 micrograms of micronuclear DNA can be obtained from 6 x 10(7) paramecia. We find that there are internal sequences in the micronuclear A gene DNA in wild type cells which are eliminated when the micronuclei develop into macronuclei. They yield unique restriction fragments for micronuclei and macronuclei. Therefore the purity of the preparations is easily monitored by probing Southern blots of restriction enzyme-digested DNA with the cloned A gene. No differences have been found between the micronuclear A gene in wild type and the d48 mutant.

Fingerprinting human chromosomes by polymerase chain reaction-mediated DNA amplification

We describe here a method for DNA fingerprinting of human chromosomes by Alu-polymerase chain reaction (PCR) amplification of DNA from monochromosomal hybrids, following digestion with restriction endonucleases. DNA digestion with restriction enzymes prior to PCR amplification reduces the total number of amplified fragments. The number and pattern of bands of PCR products observed in an electrophoretic medium are chromosome specific and provide a "fingerprint signature" for individual human chromosomes. Using this approach, we have produced fingerprints for human chromosomes 2, 5, 7, 9, and 12. The applicability of this approach to chromosome identification was assessed by comparing the fingerprints obtained for two different hybrids containing chromosome 7. DNA fragments specific for the long and the short arms of human chromosome 12 have also been identified. In addition, Alu-PCR-generated DNA fragments, specific for different chromosomes, were used to probe Southern blots of a hybrid cell panel to identify human chromosomes present in hybrid cell lines. The chromosomal specificity of these probes permits the identification of intact as well as rearranged chromosomes composed of segments arising from more than one chromosome.

Determination of the nucleotide sequence and chromosomal localization of the ATP2B2 Gene Encoding Human Ca 2+-Pumping ATPase Isoform PMCA2

The plasma membrane Ca(2+)-pumping ATPase (Ca(2+)-ATPase) is responsible for maintaining calcium homeostasis in eukaryotic cells. The Ca(2+)-ATPase is a family of pumps that are encoded by at least four genes. A cDNA for the human version of Ca(2+)-ATPase isoform PMCA2 was isolated and characterized. Comparison of the human and rat cDNA sequences showed that they were 95% homologous in the coding domain, and this homology was reflected in the deduced protein sequence where greater than 98% homology between the human and rat sequences was found. The amino acid differences that were found were almost all conservative. The PMCA2 cDNA was used to probe Southern blots of human-rodent somatic cell hybrid DNAs; the results indicated that the human PMCA2 gene was located on chromosome 3.

PRESENCE OF THE MOUSE MAMMARY-TUMOR VIRUS (MMTV) POL GENE IN BREAST-CANCER

We have used the polymerase chain reaction (PCR) to detect sequences related to the mouse mammary tumor virus (MMTV) reverse transcriptase gene in DNA from breast cancer cell lines and an extensive series of breast tumors. Similar MMTV-related sequences were observed in DNA from normal tissues. The segments amplified by PCR showed over 90% homology to the nucleotide sequence of the MMTV pol gene and no significant differences were noted between the DNA from normal or tumor tissue. When the amplified DNA was used to probe Southern blots, a unique restriction fragment indicative of a single copy locus was detected in all DNAs tested, but the high background of hybridization suggested that many closely related sequences may occur in the human genome.

Cloning and linkage analysis of Neisseria gonorrhoeae DNA methyltransferases.

We have cloned DNA methyltransferases (MTases) from various strains of Neisseria gonorrhoeae. Each of these clones represents a single specificity, indicating that the multiple gonococcal MTase specificities are encoded by monospecific MTases. The DNAs of five strains (FA5100, F62, MS11, Pgh3-2, and WR302) were digested with NheI, SpeI, or NheI plus SpeI and subjected to pulsed-field gel electrophoresis. The DNA MTase clones were used to probe Southern blots of these pulsed-field gels to determine whether the MTase genes are linked and whether there are strain-to-strain differences. The results indicate that none of these genes are closely linked, but variable hybridization patterns indicate that there exist restriction fragment length polymorphisms between the strains tested. Most of the chromosomal regions containing these restriction fragment length polymorphisms are clustered in regions containing gonococcal genes known or suspected to antigenically vary via genetic recombination.

A DNA methylation imprint, determined by the sex of the parent, distinguishes the angelman and Prader-Willi syndromes

The Angelman (AS) and Prader-Willi (PWS) syndromes are two clinically distinct disorders that are caused by a differential parental origin of chromosome 15q11–q13 deletions. Both also can result from uniparenta disomy (the inheritance of both copies of chromosome 15 from only one parent). Loss of the paternal copy of 15q11–q13, whether by deletion or maternal uniparental disomy, leads to PWS, whereas a maternal deletion or paternal uniparental disomy leads to AS. The differential modification in expression of certain mammalian genes dependent upon parental origin is knows as genomic imprinting, and AS and PWS represent the best examples of this phenomenon in humans. Although the molecular mechanisms of genomic imprinting are unknown, DNA methylation has been postulated to play a role in the imprinting process. Using restriction digests with the methyl-sensitive enzymes HpaII and HhaI and probing Southern blots with several genomic and cDNA probes, we have systematically scanned segments of 15q11–q13 for DNA methylation differences between patients with PWS (20 deletion, 20 uniparental disomy) and those with AS (26 deletion, 1 uniparental disomy). The highly evolutionarily conserved cDNA, DN34, identifies distinct differences in DNA methylation of the parental alleles at the D15S9 locus. Thus, DNA methylation may be used as a reliable, posnatal diagnostic tool in these syndromes. Furthermore, our findings demonstrate the first known epigenetic event, dependent on the sex of the parent, for a locus within 15q11–q13. We propose that expression of the gene detected by DN34 is regulated by genomic imprinting and, therefore, that it is a candidate gene for PWS and/or AS.

Phylogenetic Relationships of the Sweetpotato [Ipomoea batatas (L.) Lam.

Twenty-four accessions of Ipomoea, representing 13 species of section Batatas and the outgroup species I. gracilis and I. pes-caprae were analyzed for restriction fragment length polymorphisms. Polymorphisms were detected by probing Southern blots of restriction enzyme-digested genomic DNA with 20 low or moderate copy number sequences isolated from an I. batatas cv. Georgia Red genomic library. Data were analyzed cladistically and phenetically. Ipomoea trifida, I. tabascana, and collection K233 are, of the materials examined, the most closely related to sweetpotato (I. batatas). Ipomoea littoralis, the only Old World species in the section, is a sister species to I. tiliacea. Ipomoea littoralis, I. umbraticola, I. peruviana, I. cynanchifolia, and I. gracilis are shown to be diploid (2n = 2x = 30). In contrast, I. tabascana is tetraploid (2n = 4x = 60). The intrasectional relationships of section Batatas species and the role of tetraploid related species in the evolution of the cultivated I. batatas are discussed.

Localization of the human UBC polyubiquitin gene to chromosome band 12q24.3

The localization of specific human ubiquitin genes has not been straightforward because of the conservation of the ubiquitin coding sequence and the number of processed pseudogenes. An ⋍1.4-kb sequence from the 5′-flanking region of the UBC gene has been shown to be unique to that locus and free from dispersed repeat elements. The cloned 5′-flanking fragment has been used to probe Southern blots of DNA obtained from somatic cell hybrid cell lines. These data indicate that the UBC gene is located on chromosome 12. In situ hybridization with the 5′-flanking probe has refined the assignment to the broad chromosomal subband 12q24.3. These data show that the active ubiquitin genes are not clustered and are located on separate chromosomes. In addition, these studies demonstrate the utility of intron or flanking sequence probes in the specific chromosomal assignment of members of highly conserved gene families.

A new, sensitive polynucleotide probe for distinguishingCandida albicansstrains and its use with a computer assisted archiving and pattern comparison system

The repetitive DNA sequence poly[d(GT).d(CA)],(polyGT) can be used to generate DNA fingerprints that distinguish different yeast genera. In this study we demonstrate that the probe can also be used to distinguish individual strains of clinical isolates of Candida albicans. Isolates were fingerprinted by probing Southern blots of restriction enzyme-cleaved DNA samples with radioactively labelled polyGT. The discrimination between strains was clearer than can be achieved by direct visualization of ethidium bromide stained gels and was comparable to that achieved with previous DNA probes. However, the advantage of this probe is that it is not limited to Candida species since polyGT sequences appear to be ubiquitous in eukaryotes. Fingerprints were also generated from Southern blots using a commercial (AMBIS) radioanalytical imaging computer system. A proprietary software package (MICRO PM) was effective in discriminating between the C. albicans strains. These preliminary results indicate the potential value of this probe for discrimination between Candida isolates in epidemiological studies of candidosis.

The aconitase of Escherichia coli: purification of the enzyme and molecular cloning and map location of the gene (acn)

The aconitase of Escherichia coli was purified to homogeneity, albeit in low yield (0.6%). It was shown to be a monomeric protein of Mr 95,000 or 97,500 by gel filtration and SDS-PAGE analysis, respectively. The N-terminal amino acid sequence resembled that of the Bacillus subtilis enzyme (citB product), but the similarity at the DNA level was insufficient to allow detection of the E. coli acn gene using a 456 bp citB probe. Phages containing the acn gene were isolated from a lambda-E. coli gene bank by immunoscreening with an antiserum raised against purified bacterial enzyme. The acn gene was located at 28 min (1350 kb) in the physical map of the E. coli chromosome by probing Southern blots with a fragment of the gene. Attempts to locate the gene using the same procedure with oligonucleotide probes encoding segments of the N-terminal amino acid sequence were complicated by the lack of probe specificity and an inaccuracy in the physical map of Kohara et al. (Cell 50, 495-508, 1987). Aconitase specific activity was amplified some 20-200-fold in cultures transformed with pGS447, a derivative of pUC119 containing the acn gene, and an apparent four-fold activation-deactivation of the phagemid-encoded enzyme was observed in late exponential phase. The aconitase antiserum cross-reacted with both the porcine and Salmonella typhimurium (Mr 120,000) enzymes.

Differentiation of Fusarium solani f. sp. cucurbitae races 1 and 2 by random amplification of polymorphic DNA

We have used a PCR-based technique, involving the random amplification of polymorphic DNA (RAPD), to assess genome variability between 21 isolates from F. solani f. sp. cucurbitae races 1 and 2. Based on RAPD marker patterns the isolates fell into two distinct groups corresponding to mating populations MPI and MPV. Four isolates that could not be assigned to one or other mating population by traditional means were distinguished by RAPD patterns. Seven polymorphic RAPD products were used to probe Southern blots of MPI and MPV genomic DNA. Six of the seven probes hybridized to single-copy sequences and five of the seven probes showed specificity for one or other mating population. We suggest that not only is the technique a rapid and reliable tool for isolate-typing of fungi but it also provides a rapid method for obtaining species- or race-specific hybridization probes.

Association and linkage analyses of restriction fragment length polymorphisms for the human renin and antithrombin III genes in essential hypertension

Essential hypertension is a highly hereditable disorder in which genetic influences predominate over environmental factors. The molecular genetic profiles which predispose to essential hypertension are not known. In rats with genetic hypertension, there is some recent evidence pointing to linkage of renin gene alleles with blood pressure. The genes for renin and antithrombin III belong to a conserved synteny group which, in humans, spans the q21.3-32.3 region of chromosome I and, in rats, is linkage group X on chromosome 13. The present study examined the association of particular human renin gene (REN) and antithrombin III gene (AT3) polymorphisms with essential hypertension by comparing the frequency of specific alleles for each of these genes in 50 hypertensive offspring of hypertensive parents and 91 normotensive offspring of normotensive parents. In addition, linkage relationships were examined in hypertensive pedigrees with multiple affected individuals. Alleles of a REN HindIII restriction fragment length polymorphism (RFLP) were detected using a genomic clone, lambda HR5, to probe Southern blots of HindIII-cut leucocyte DNA, and those for an AT3 PstI RFLP were detected by phATIII 113 complementary DNA probe. The frequencies of each REN allele in the hypertensive group were 0.76 and 0.24 compared with 0.74 and 0.26 in the normotensive group. For AT3, hypertensive allele frequencies were 0.49 and 0.51 compared with normotensive values of 0.54 and 0.46. These differences were not significant by chi 2 analysis (P greater than 0.2).(ABSTRACT TRUNCATED AT 250 WORDS)