Southern Blot Hybridization Of DNA Research Articles

Prevalence and molecular characteristics of mcr-1 colistin resistance in Escherichia coli: isolates of clinical infection from a Chinese University Hospital.

BackgroundColistin has been considered as one of the most effective treatments in clinical infections, especially multidrug-resistant (MDR) bacteria-infected patients. The mcr-1 gene leads to polymyxin resistance in China. The present study investigated the prevalence of mcr-1 in a Chinese teaching hospital, and the molecular phenotypes of the positive strains were analyzed.MethodsA total of 1,112 Escherichia coli strains were collected from a Chinese University Hospital from January 2015 to January 2016. The mcr-1 gene was detected by PCR. All positive specimens were subjected to susceptibility testing, clinical analysis, phylogenetic analysis, DNA Southern blot hybridization, and gene sequencing.ResultsSix (0.6%) strains of mcr-1-positive E. coli were susceptible to imipenem, meropenem, and tigecycline, except for one that presented moderate levels of tigecycline resistance. The six isolates were resistant to cefotaxime and cefepime and divided into six types of sequences. These positive strains carried a total of three plasmids: approximately 33, 61, and >92 kb plasmids. All patients were eventually cured using different types of antibiotics and discharged.ConclusionThe current study showed that the mcr-1 gene was responsible for the majority of colistin resistance in clinical isolates of E. coli. The gene can be transferred into plasmids containing other drug resistance genes by plasmid–DNA conjugation, which might cause severe consequences in drug-resistant strains. Thus, the widespread popularity of mcr-1 gene should be prevented.

Survey of the Distribution of Different Types of Nitrogenases and Hydrogenases in Heterocyst-Forming Cyanobactera

As a first step toward developing the methodology for screening large numbers of heterocyst-forming freshwater cyanobacteria strains for the presence of various types of nitrogenases and hydrogenases, we surveyed the distribution of these genes and their activities in 14 strains from culture collections. The nitrogenase genes include nif1 encoding a Mo-type nitrogenase expressed in heterocysts, nif2 expressed in vegetative cells and heterocysts under anaerobic conditions, and vnf encoding a V-type nitrogenase expressed in heterocysts. Two methods proved to be valuable in surveying the distribution of nitrogenase types. The first method was Southern blot hybridization of DNA digested with two different endonucleases and hybridized with nifD1, nifD2, and vnfD probes. The second method was ethane formation from acetylene to detect the presence of active V-nitrogenase. We found that all 14 strains have nifD1 genes, and eight strains also have nifD2 genes. Four of the strains have vnfD genes, in addition to nifD2 genes. It is curious that three of these four strains had similar hybridization patterns with all of the nifD1, nifD2, and vnfD probes, suggesting that there could be some bias in strains used in the present study or in strains held in culture collections. This point will need to be assessed in the future. For surveying the distribution of hydrogenases, Southern blot hybridization was an effective method. All strains surveyed had hup genes, with the majority of them also having hox genes.

Telomerase activity, MIB-1, PCNA, HPV 16 and p53 as diagnostic markers for cervical intraepithelial neoplasia.

The present investigation evaluated the relationship between dysplasia of the uterine cervix and telomerase activity, expression of p53, MIB-1 and PCNA. Telomerase activity was measured on cervical cytobrush material from 126 women suspected of having dysplasia and 61 controls using the telomeric repeat amplification protocol. Immunohistochemistry was used to detect the tumor suppressor protein p53 and cell proliferation, the latter by MIB-1 and PCNA expression. Infection with human papillomavirus 16 was detected by PCR amplification and Southern blot hybridization of DNA extracted from the same brush material. Positive telomerase activity was found in 5 of 43 (11.6%) normal samples, 12 of 57 (21.1%) samples with inflammation or koilocytosis, 7 of 17 (41.2%) CIN 1 (cervical intraepithelial neoplasia, grade 1), 8 of 20 (40.0%) CIN 2, and 25 of 42 (59.5%) CIN 3/ CIS. Telomerase activity was significantly related to the level of dysplasia (p<0.001) and proliferation measured by MIB-1 (p=0.019), but not to the level of PCNA (p=0.445), HPV 16 status (p=0.098) or staining for p53 (p=0.271). Dysplasia was also related to PCNA, MIB1, p53, and presence of HPV 16. A sequential increase in the examined parameters, paralleling the progression of abnormality, was observed. PCNA and telomerase showed an increase in CIN 1, MIB-1 and HPV16 in CIN 2, and finally p53 in CIN 3/CIS.

Bleomycin resistance in Staphylococcus aureus clinical isolates

Among clinical isolates of Staphylococcus aureus tested for resistance to the antibiotic bleomycin, 197 were found to be resistant; most of them were also resistant to tobramycin and contained plasmids. DNA dot-blot hybridization analysis of the bleomycin resistant isolates with an 171 bp probe derived from the plasmid pUB110 indicated that 43 strains (22%) carried pUB110-like bleomycin resistance DNA sequences. Analysis of bacterial cell lysates derived from the bleomycin resistant isolates indicated that many contained bleomycin-binding properties (BBP), that prevent DNA damage by the antibiotic. Of 13 strains that were analysed by DNA gel electrophoresis and Southern blot DNA hybridization, six were found to carry pUB110-like bleomycin resistance DNA sequences. These studies indicate that there may be more than one genetic determinant for bleomycin resistance in S. aureus whose DNA is protected.

Conservation of the cytotoxin-associated (cagA) gene ofHelicobacter pyloriand investigation of association with vacuolating-cytotoxin activity and gastroduodenal disease

Polymerase chain reaction (PCR) amplification and DNA hybridization analyses were used to test for the presence of the cytotoxin-associated (cagA) gene in 108 strains of Helicobacter pylori. Fifty-two geographically diverse strains of known vacuolating cytotoxin activity, and 56 recent UK clinical isolates from patients with duodenal ulceration (n = 28) and from healthy individuals who were endoscopically normal (n = 28) were studied. Overall, cagA was detected by PCR in 74 (69%) strains and DNA hybridization provided evidence of gene homologues in a further eight strains. For 96% of the cytotoxin-producing strains and 46% of the non-cytotoxin producing strains, there was a close association either with presence or absence of cagA. At the genomic level, Southern blot DNA hybridization showed that cagA was probably present in a single copy in most of the H. pylori tested, and that HaeIII restriction site variation within and around the gene provided additional markers of diversity for the species. As 40% of the cagA containing strains did not produce an active cytotoxin, and no significant association between cagA presence and DU-disease was observed, we concluded that the presence of the cagA gene in H. pylori could not be used as a single reliable predictor of higher risk patients.

Human Zn-α2-glycoprotein: Complete genomic sequence, identification of a related pseudogene and relationship to class I major histocompatibility complex genes

The human gene (AZGPI) encoding Zn-α2-glycoprotein(Zn-α2-gp), a protein present in several biological fluids and produced by a subtype of breast carcinomas, has been cloned and its complete nucleotide sequence determined. The gene spans over 9.7 kb, and its overall organization and nucleotide sequence are very similar to those of the first four exons of class I MHC genes. However, the Zn-α2-gp gene differs from these genes in several significant ways. It lacks the coding information for the transmembrane and cytoplasmic domains typical of MHC genes, which is consistent with its presence as a soluble protein in different physiological and pathological fluids. In addition, it contains a high density of repetitive sequences, including Alu, MER, and MIR elements, which are not present at equivalent positions in class I MHC genes. Finally, its 5′-flanking region lacks the class I MHC regulatory complex and the interferon consensus sequence characteristic of class I MHC genes. These findings may explain the different expression pattern of Zn-α2-gp and class I MHC genes in human tissues. Southern blot hybridization of DNA from several species with a cDNA probe indicated that Zn-α2-gp genes are present in a wide variety of animal species, including monkey, rat, mouse, dog, cow, and rabbit. The human genome also contains a putative Zn-α2-gp pseudogene that has been isolated and partially characterized. This pseudogene has an intron—exon organization identical to that of the functional gene, but it presents two deleterious mutations in the third exon that lead to the appearance of premature stop codons. Finally, considering the lack of polymorphism in the Zn-α2-gp gene in comparison with MHC genes, putative roles for this human glycoprotein in the transport of nonpolymorphic substances or in intercellular recognition processes are proposed.

Human 25-hydroxyvitamin D 24-hydroxylase cytochrome P450 subunit maps to a different chromosomal location than that of pseudovitamin D-deficient rickets.

We have cloned part of the human 25-OHD 24-hydroxylase cytochrome P450 (P450cc24) cDNA. The characterized sequence consists of 776 bp of the coding and 720 bp of the 3'-untranslated region interrupted by an intron. In the coding region we found 79.8% similarity in DNA and 87.5% in deduced amino acid sequences between human and rat, with no similarity in the 3'-untranslated region. By Southern blot hybridization of DNA from human-hamster somatic cell hybrids and by in situ immunofluorescence hybridization, we mapped P450cc24 to human chromosome 20q13.1. This location of P450cc24 is different from that of pseudovitamin D-deficient rickets (PDDR), previously assigned to chromosome 12q14 by linkage analysis, thus excluding it as a target of the PDDR mutation. Since it is likely that PDDR is caused by a mutation in the 25-OHD 1 alpha-hydroxylase P450 subunit (P450cc1 alpha) our results do not support the hypothesis that the two cytochromes are encoded by a single gene.

Benign lymphocytic angiitis and granulomatosis: A T-cell lymphoma?

Benign lymphocytic angiitis and granulomatosis is a T-cell lymphoproliferative disorder confined to the lung and corresponding to a low-grade angiocentric immunoproliferative lesion. Controversy remains as to whether these lesions are lymphomas. We report such a case in an 8-year-old patient with Burkitt's lymphoma in remission who presented with persistent bronchopneumopathy and bilateral pulmonary infiltrates on tomodensitometry. Surgical resection revealed the histologic changes of benign lymphocylic angiitis and granulomatosis. Immunohistochemistry showed no aberrant pan T-cell marker loss. Genetic analysis of frozen tissue by Southern blot DNA hybridization with probes to T-cell receptor beta- and gamma-chain genes and to the immunoglobulin heavy chain joining region gene (JH) identified no clonal rearrangement. Search for Epstein-Barr virus-DNA sequences by in situ hybridization and Southern blot analysis provided negative results. Our data imply that low-grade angiocentric immunoproliferative lesions are not exclusively lymphomas but might represent a borderline lymphoproliferative disease (seen in the course of many diseases), perhaps corresponding to host immune response.

Isolation and characterization of two temperate phages from Lactococcus lactis ssp. cremoris C3

Lactococcus lactis ssp. cremoris C3 was shown to contain two prophages, C3-T1 and C3-T2, both inducible bymitomycin C. The phages were morphologically indistinguishable, having an isometric head (55 nm), a noncontractile tail (142 nm), and a distinctive base plate. The phages C3-T1 and C3-T2 were differentiated from one another by restriction endonuclease analysis of their DNA, and genome sizes of 37.8 (C3-T1) and 32.5 kb (C3-T2). Southern blot DNA hybridization suggested a maximum homology between the phage genomes of 50%. Phage C3-T1 was propagated and the phage obtained was designated C3-TIℓ. Phage C3-T2 was not propagated despite tests with a large number of possible indicator strains. Restriction enzyme analysis of phage C3-T1ℓ genome yielded a 37.8-kb circular restriction map. The packaging site (pac) and site of integration into the bacterial chromosome (att) were located and an EcoRI DNA fragment containing the att site was cloned. Only one C3-T1 att site was present in the C3 chromosome. No homology was detected between DNA from C3-T1ℓ and DNA from lytic phages commonly isolated from cheese wheys. Key words: lactococcal phages, temperate phages, lysogeny, evolution.

Chromosomal localization of a cytochrome b 5 gene to human chromosome 18 and a cytochrome b 5 pseudogene to the X chromosome

We have isolated cDNA clones that code for human cytochrome b 5. Owing to the high degree of evolutionary conservation of cytochrome b 5 sequences and the existence of human and rodent cytochrome b 5 processed pseudogenes, we were unable to map unambiguously the chromosomal localization of the human gene(s) by Southern blot hybridization of DNA from human-rodent somatic cell hybrids. An alternative approach, based on restriction enzyme digestion of PCR-amplified DNA, enabled us to map the human cytochrome b 5 gene(s) to chromosome 18 and one of its processed pseudogenes to the X chromosome. We propose the designations CYB5 and CYB5P1 for the gene and pseudogene loci, respectively.

Is vestibular papillomatosis associated with human papillomavirus?

The origin and clinical significance of vestibular papillae were evaluated by comparing histological features with the presence of human papillomavirus (HPV) types 6/11 and 16/18, as revealed by Southern blot DNA hybridization. Twenty women with vestibular papillomatosis underwent clinical evaluation and follow-up. When available, male partners were also examined. Histological changes suggestive of HPV infection were present in all the 20 specimens. Sixteen cases (80%) contained DNA sequences homologous to the viral probes. In particular, 12 cases (60%) reacted with the HPV 16/18 probe. Follow-up for more than 18 months revealed no variation in the distribution and appearance of vestibular papillae. No male partner showed signs of HPV lesions. The study shows that HPV 16 is frequently associated with vestibular papillae but does not support a productive infection. Therefore the most appropriate management of these patients should be evaluated clinically in each individual case.

Clonal Immunoglobulin Gene Rearrangement in the Infarcted Lymph Node Syndrome

The authors report a case of complete lymph node infarction in which a specific etiology could not be determined by morphologic or immunophenotypic studies; however, clonal rearrangement of the immunoglobulin gene was demonstrated by Southern blot hybridization of DNA extracted from the necrotic tissue. A subsequent lymph node biopsy later was diagnosed as malignant lymphoma, using morphologic, immunophenotypic and genotypic criteria. Identical clonally rearranged bands were present in DNA from both the infarcted nodal and the subsequent tissue biopsies. In the setting of lymph node necrosis, gene rearrangement studies may provide diagnostic information concerning clonality, even if morphologic and immunophenotypic studies are indeterminate for a lymphoproliferative process.

A case of classical mycosis fungoides associated with human T-cell lymphotropic virus type I

A 72-year-old male patient from north-eastern Iran developed the typical clinical and histopathological features of mycosis fungoides with lymphadenopathy, but without any other systemic involvement. Human T-cell lymphotropic virus (HTLV-I) antibodies were detected in the patient's serum by two different ELISAs and by Western blot using purified viral particles from MT-2 culture supernatants. Cultured peripheral blood lymphocytes were positive for labelling with anti-HTLV-I serum. Southern blot hybridization of DNA extracted from a skin tumour and from an involved lymph node revealed integrated proviral DNA with identical restriction patterns. This case supports a relationship between mycosis fungoides and HTLV-I and may indicate a new region of endemic HTLV-I infection.

Chromosomal localization ofCd1d genes in the mouse

Southern blot hybridization of DNA from Chinese hamster x mouse somatic cell hybrids was used to assign the mouse Cd1d genes to chromosome 3. Analysis of the progeny of an intersubspecies backcross was used to position these genes near the gene for glucocerebrosidase, Gba.

Detection of Carriers for X-linked Ichthyosis by Southern Blot Analysis and Identification of One Family with a De Novo Mutation

Scaling in patients with recessive X-linked ichthyosis is caused by lack of activity of the enzyme steroid sulfatase. In approximately 90% of kindreds, this lack is the result of a DNA deletion large enough to eliminate the coding region completely. We have used Southern blot hybridization of DNA isolated from peripheral blood leukocytes to measure gene dosage of the steroid sulfatase gene. This readily detects a half-normal dosage in women who are carriers and therefore can be used to diagnose the carrier status of female relatives of 90% of patients with the disease. We have found one family in whom the deletion arose on an allele inherited from the proband's clinically normal maternal grandfather.

Genomic arrangement of repeated PS700 elements in the nematode Panagrellus silusiae

When genomic DNA from the free-living nematode Panagrellus silusiae is digested with the restriction endonuclease BamHI and separated by electrophoresis, a band in the 700 base pair size range is evident after ethidium bromide staining. One of the 0.7-kilobase fragments (PS700-1) was characterized and found to be a member of a moderately repetitive DNA family (T. Warren and J.J. Pasternak. 1988. Nucleic Acids Res. 16: 10,833-10,847). In the current study, DNA sequence analyses of three independently isolated copies of the PS700 DNA family showed the same nucleotide sequence and greater than 98% similarity to PS700-1. Four EMBL-4 bacteriophage clones were isolated from a Panagrellus genomic DNA library with PS700-1 as the probe and were analyzed by restriction endonuclease site mapping and Southern blot DNA hybridization. These clones contain 31 copies of the PS700 DNA family. In each case, the units are arranged in head-to-tail arrays. One of the EMBL-4 clones contains copies of a novel variant of the PS700 elements. The maintenance of both nucleotide sequence and restriction endonuclease restriction site homogeneity among members of the dispersed PS700 DNA family may denote a functional role for these sequences.

Detection of Epstein‐Barr Virus DNA in Reed‐Sternberg Cells of Hodgkin's Disease Using the Polymerase Chain Reaction and in situ Hybridization

Thirty‐one cases of Hodgkin's disease were examined for the occurrence of Epstein‐Barr virus (EBV) genome by using the polymerase chain reaction (PCR) of DNA in formalin‐fixed paraffin‐embedded tissues and the in situ hybridization technique. The cases were subdivided into 17 cases of nodular sclerosis (NS), nine cases of mixed cellularity (MC), four cases of lymphocyte predominance (LP), and one case of lymphocyte depletion (LD). EBV DNA was detected in eight cases including four cases of NS, three cases of MC and one case of LP. The sensitivity of PCR was higher than that of Southern blot hybridization of DNA from fresh frozen tissue, because Southern blot hybridization using the BamHI‐W fragment of EBV detected virus DNA only in two of three cases which were positive by PCR. The results of in situ hybridization studies confirmed that EBV genome was localized within the nuclei of Reed‐Sternberg (RS) cells and their mononuclear variants. Furthermore, double‐labeling studies combining in situ hybridization and immunocytochemistry using CD30 (BerH2) and CD15 (LeuM1) as markers of RS cells, as well as pan B‐marker (L26) and pan T‐marker, CD45RO (UCHL1), were performed to demonstrate the phenotype of EBV DNA‐positive cells, confirming that EBV DNA was present in RS cells but not in lymphocytes. The results of this study indicate a significant association between EBV and some cases of Hodgkin's disease.

Transformation of the entomopathogenic fungusMetarhizium anisopliaeto benomyl resistance

Protoplasts of the entomopathogenic fungus Metarhizium anisopliae were transformed to benomyl resistance using cosmid pSV50 which harbours a β-tubulin gene cloned from a Neurospora crassa benomyl-resistant mutant. Transformant colonies, which appeared at a frequency of 4 per 50 μg DNA, grew and sporulated on 10 μg/ml benomyl, whereas the wild type was inhibited by 3 μg/ml. Southern blot hybridization of DNA from transformants showed that, in each case, tandem repeats of the cosmid had integrated at several chromosomal loci. The transformants were mitotically stable when subcultured on non-selective agar and retained the ability to infect and kill larvae of Manduca sexta. Two transformants were less virulent than the wild type and one of them showed slower in vitro spore germination. The benomyl-resistant phenotype persisted in reisolates from insect cadavers.

Resolution of vaccinia virus DNA concatemer junctions requires late-gene expression.

Vaccinia virus replicates in the cytoplasm of infected cells, generating transient replicative intermediates containing the DNA for the terminal sequences as concatemeric junctions. The processing of the terminal sequences for a series of vaccinia virus conditional lethal mutants at the nonpermissive temperature was analyzed by restriction enzyme digestion and Southern blot hybridization of DNA isolated from infected cells. Three phenotypes were observed: DNA replication negative (Rep-), DNA replication positive but concatemer resolution negative (Rep+ Res-), and DNA replication positive and concatemer resolution positive (Rep+ Res+). Interestingly, all six Rep+ Res- mutants from separate complementation groups were defective in late protein synthesis. Isatin beta-thiosemicarbazone, a drug that blocks late protein synthesis, also prevented resolution of concatemers. Orthogonal field gel electrophoresis of the DNA generated by the late defective mutants revealed a distribution of linear genome multimers. The multimers were processed into mature monomers after a shift to the permissive temperature in the presence of cytosine arabinoside for all the Rep+ Res- mutants except ts22, an irreversible mutant which cleaves RNA late in infection (R.F. Pacha and R.C. Condit, J. Virol. 56:395-403, 1985). Genome formation can be divided into two stages: DNA replication, which generates concatemers, and resolution, which processes concatemers into monomers with hairpin termini. Early viral genes are required for the former, and late viral genes are required for the latter.

Warthin’s Tumor with Malignant Lymphoma: DNA Analysis of Paraffin-Embedded Tissue

The authors report a case of malignant lymphoma, small lymphocytic type, involving the lymphoid stroma of a Warthin's tumor of the parotid gland. This was confirmed by the presence of a monoclonal immunoglobulin heavy chain gene rearrangement, demonstrated by Southern blot hybridization of DNA extracted from paraffin-embedded tissue. Similar techniques showed only germline immunoglobulin gene bands in two control cases of Warthin's tumor.