Southern Blot Hybridization Analysis Research Articles

Spatial and temporal expression of 4f-rnp gene in Drosophila melanogaster

We have recently discovered that a pre-messenger RNA (mRNA) encoded by the 4f-rnp gene in Drosophila melanogaster undergoes RNA editing by site-specific A-to-G conversions. In this report, we describe the spatial and temporal expression patterns of 4f-rnp mRNA transcripts during fly development. The 4f-rnp locus was mapped by polytene in situ hybridization experiments to the 4F1,2 bands at the distal tip of the X-chromosome, demonstrating that this gene is nuclear. We show that 4f-rnp is a single-copy gene by a combination of genomic Southern blot hybridization and restriction map analysis of clones isolated through chromosome walking techniques. Northern blot analysis indicates that two alternatively spliced messenger RNA transcripts, differing in length by 200–300 nucleotides, are synthesized in adult male and female flies, during embryogenesis and throughout the larval period. Using tissue in situ hybridization techniques that do not distinguish between the transcripts, we find that the distribution of 4f-rnp mRNAs is specific to germline tissue in the ovary, demonstrating that these transcripts are maternally produced. During embryogenesis, 4f-rnp transcripts are localized within cells of most tissues, but they have a very distinct localization pattern within the segmental ganglia of the central nervous system. We discuss the significance that RNA editing is expected to have for the predicted 4F-RNP protein products that may be expressed throughout fly development and in high abundance within the fly's central nervous system.

The course of disease and persistence of virus in the central nervous system varies between individual CBA mice infected with the BeAn strain of Theiler's murine encephalomyelitis virus.

Following intracerebral inoculation of the BeAn strain of Theiler's murine encephalomyelitis virus the course of the acute infection and persistence of virus in the CNS varies between individual CBA mice. On the basis of clinical signs, virus distribution, virus titres, histopathology and Southern blot hybridization analysis of virus specific RT-PCR amplified products from total brain and spinal cord RNA, individual CBA mice could be placed into one of three groups. The first group were those animals which died of acute encephalitis. The second group were animals with or without clinical signs which had early high CNS virus titres, and in addition to scattered foci of infection had spread of virus in specific neuronal nuclei followed by destruction of these areas and thereafter persistence of virus in the CNS. The third group had no clinical signs, low CNS virus titres, small foci of CNS infection and were negative for virus after 28 days. This third pattern of infection was also seen in BALB/c mice. Between 50 and 268 days post-infection 53% of CBA mice were positive for viral RNA in the CNS by RT-PCR. No BALB/c mice were positive. In both the acute and persistent infection there was a correlation between serum neutralizing antibody titre and clinical signs. During the acute infection BeAn RNA could be detected in neurons and astrocytes. Oligodendrocytes were negative. In those CBA mice with persistence, viral RNA was observed in scattered individual or small foci of cells, predominantly oligodendrocytes, in both the brain and spinal cord.

The enterohemolysin phenotype of bovine Shiga-like toxin-producing Escherichia coli (SLTEC) is encoded by the EHEC-hemolysin gene

Naturally occurring enterohemolysin negative variants were observed during studies on bovine Shiga-like toxin-producing E. coli (SLTEC). Examination of three strains ( 413 89-1 and 332, O26:H-, and 570 89 , O111:H-) and their isogenic variants ( 413 89-6 , 332-I and 570 89-I , respectively) showed, that in each strain loss of the enterohemolytic phenotype correlated with the loss of a large plasmid ranging from 94 to 104 kb in size. The hemolysin determinant present on the 94 kb plasmid of strain 413 89-1 was cloned and discovered by DNA and N-terminal aminoacid sequence analysis to be highly homologous to the recently published EHEC-hemolysin (Hly EHEC; Schmidt et al., 1994; 1995). When a recombinant plasmid harboring this determinant was reintroduced into the enterohemolysin negative isogenic mutant 413 89-6 , the enterohemolytic phenotype was restored. Southern blot hybridization analysis was used to demonstrate that the Hly EHEC is plasmid-borne in SLTEC-strains. Our cumulative data suggest that the enterohemolytic phenotype of SLTEC is encoded by the plasmid-borne Hly EHEC. These results further demonstrate the close similarity between SLTEC-isolates from bovine and human.

A third Ig light chain gene isotype in Xenopus laevis consists of six distinct VL families and is related to mammalian lambda genes.

Xenopus laevis is a unique model for studying the ontogenetic development of immune functions. A short primer PCR amplification method was employed to amplify fragments from Xenopus genomic DNA that are related to Ig light chains and TCR. One fragment was identified that appeared to represent a novel type of light chain and was used as a probe to recover the corresponding cDNA from a spleen cDNA library. We designate these light chains type III. Using an iterative screening procedure, six families of VL genes, two distinct JL and two distinct CL sequences, were identified. In a comparison of phylogenetically diverse light chains, the type III genes align with higher vertebrate lambda genes. Southern blot hybridization analyses with genomic DNA from different animals showed the VL and CL sequences to be both diverse and polymorphic. Intrafamily sequence comparisons of VL genes revealed additional diversity. Collectively, these studies confirm the existence of a third type of light chain gene in Xenopus, establish a high degree of genetic variation in the sequences encoding the light chain V regions, and provide the most significant evidence to date for the presence of a lambda-like light chain gene at the phylogenetic level of the amphibians.

Expression of c-fos and c-jun proto-oncogenes by ovine preimplantation embryos.

The c-fos and c-jun proto-oncogenes are involved in the regulation of gene expression, cell proliferation, differentiation and tumorigenesis. Embryogenesis, like tumorigenesis, involves dramatic cell growth, cleavage and differentiation processes. Activation of the c-fos and c-jun proto-oncogenes in sheep conceptuses during the period of rapid growth and elongation was examined using reverse transcription and polymerase chain reaction (RT-PCR). The specificity of PCR products was determined by Southern blot hybridisation analysis with a non-radioactive DNA probe. A band corresponding to a 507 bp fragment (predicted amplified c-fos gene cDNA product) was observed in 3 of 9 day-13, 1 of 4 day-14 and 1 of 2 day-16 embryos. Meanwhile, a 400 bp c-jun transcript was also detected in 1 of 2 day-12, 3 of 9 day-13 and 2 of 2 day-16 embryos. These results suggest that mRNA transcripts of c-fos and c-jun proto-oncogenes were specifically expressed during the period of ovine embryonic elongation and may have a possible role in preimplantation embryonic development of sheep.

Isolation and propagation of yolk-sac-derived endothelial cells from a hypervascular transgenic mouse expressing a gain-of-function fps/fes proto-oncogene.

We report on the isolation and propagation of endothelial cells from the mouse embryonic yolk sac, the earliest site of blood vessel development, and on the advantages of a hypervascular transgenic mouse source of these cells. These transgenic mice express multiple copies of an activated allele of the human fps/fes proto-oncogene and display hypervascularity progressing to multifocal hemangiomas. This phenotype suggested a role of the fps/fes proto-oncogene in vasculogenesis and angiogenesis and led us to investigate the growth characteristics of yolk-sac-derived endothelial cells from transgenic fps/fes embryos. We have established eight independent cell clones from a mixture of transgenic and control yolk sacs from Day 12 embryos. Southern blot hybridization analysis showed all eight clones to be derived from transgenic cells, suggesting a growth advantage of cells carrying the activated fps/fes gene. A cell line, Clone 166 (C166), established from one of these clones, was more fully characterized. C166 exhibits normal endothelial characteristics, such as rearrangement into tubelike structures when placed on Matrigel, expression of angiotensin converting enzyme, retention of cobblestone morphology at confluence, and the presence of cell surface receptors for acetylated low density lipoprotein. The cells constitutively express murine endothelial cell adhesion molecule VCAM-1 and the vascular addressin identified by antibody MECA-99. As expected, the cell line expresses high levels of the cytoplasmic protein-tyrosine kinase encoded by the fps/fes proto-oncogene. The clone we have described as well as other endothelial cell lines that we have established from the mouse embryonic yolk sac should prove useful for the study of endothelial cell differentiation and for the determination of the mechanisms underlying the establishment of organ-specific endothelial cell heterogeneity.

Pancreatic endocrine dysfunction in rats not expressing the cholecystokinin-A receptor.

Cholecystokinin (CCK) has been suggested to modulate insulin output. We have shown that Otsuka Long-Evans Tokushima Fatty (OLETF) rats show little or no expression of the CCK-A receptor gene in the pancreas. We examined whether the CCK-A and CCK-B receptor genes are expressed in the islets and the role of CCK-A receptor in insulin secretion. Gene expressions of CCK receptors were determined by the reverse-transcriptase polymerase chain reaction (RT-PCR) followed by Southern blot hybridization and Northern transfer analysis using LETO rats as controls. Pancreatic endocrine function was examined in perfusion (exogenous CCK stimulation) and meal ingestion (endogenous CCK stimulation) studies. CCK-A receptor mRNA was detected in the islets of LETO rats but not OLETF rats. Expression of the CCK-B receptor gene was detected in both strains by RT-PCR. Insulin secretion was impaired in OLETF rats, but the insulin contents of OLETF and LETO rats were not different. No abnormalities were detected histologically in either strain. These results suggest that the occurrence of pancreatic endocrine dysfunction in OLETF rats may be due to a defect in expression of the CCK-A receptor gene, not to insulin deficiency.

Amplification of the Transketolase Gene in Desensitization-resistant Mutant Y1 Mouse Adrenocortical Tumor Cells

As shown previously, mutants of the Y1 mouse adrenocortical tumor cell line that resist agonist-induced desensitization of adenylyl cyclase have elevated levels of a 68-kDa protein (designated p68), suggesting a possible relationship between p68 and the regulation of adenylyl cyclase activity. In the present study, cDNA cloning and sequencing were used to identify p68 as mouse transketolase. Cells overexpressing p68 exhibited a 17.4-fold increase in transketolase enzymatic activity relative to parental Y1 cells and a 28-fold amplification of the transketolase gene as determined by Southern blot hybridization analysis. Using fluorescent in situ hybridization analysis, the transketolase gene was mapped to mouse chromosome 16B1 and to human chromosome 3p21.2. Transketolase gene amplification was associated with telomeric fusion of the chromosome 16 pair together with the appearance of multiple copies of the transketolase gene throughout a different chromosome. The relationship between overexpression of transketolase and desensitization resistance was evaluated in somatic cell hybrids formed between a desensitization-resistant adrenal cell line and a desensitization-sensitive rat glial cell line. In these hybrids, transketolase overexpression behaved dominantly, whereas desensitization resistance behaved recessively. These results dissociate the desensitization resistance phenotype from overexpression of transketolase and suggest that desensitization resistance may have resulted from disruption of an essential regulatory gene in conjunction with the amplification event.

Spiroplasma citri Virus SpV1: Characterization of Viral Sequences Present in the Spiroplasmal Host Chromosome

Spiroplasma citri virus SpV1-R8A2B is a naked, rod-shaped virus with a circular, single-stranded DNA genome of 8273 bp. SpV1-related sequences were detected in the chromosomal DNA of all S. citri strains tested. Southern blot hybridization analyses revealed that several copies of most, if not all, SpV1 ORFs are present in the chromosome of S. citri strain R8A2. For further study of the integrated viral sequences, a genomic DNA library of S. citri R8A2 was constructed, and two cloned chromosomal DNA fragments containing SpV1 viral sequences were studied by comparison with the free viral genome of SpV1-R8A2B. One fragment seems to contain a full-length viral genome, while the other contains only parts of the viral genome. In both fragments, the left and right ends of viral sequences consist of 31-bp inverted repeat sequences, those which are facing each other at nucleotide 4737 in the circular viral genome. In addition, both fragments contain the SpV1-ORF3, encoding a putative transposase, immediately upstream of the right repeat. These data suggest that the SpV1-ORF3 and the repeat sequences could be parts of an IS-like element of chromosomal origin.

Ribotypes and AP‐PCR fingerprints of thermophilic campylobacters from marine recreational waters

Thirty-two strains of thermophilic campylobacters isolated from marine recreational water and seven reference strains were biotyped and analysed by chromosomal DNA HaeIII ribopatterns and AP-PCR profiles based on a random 10-mer primer (5'-CAA TCG CCG T-3'). The majority of seawater isolates (90%) were Campylobacter coli, and three strains were Camp. jejuni. Southern blot hybridization analysis showed differences between the strains, and in a numerical analysis three main clusters were formed at the 45% similarity level, that corresponded to Camp. jejuni subsp. jejuni, Camp. coli, and a combination of Camp. coli and Camp. jejuni subsp. doylei. AP-PCR profiles also differentiated between the species but were less discriminatory than ribotyping because six strains (17%) could not be typed by this method. Numerical analysis gave four main clusters at the 45% similarity level, corresponding to Camp. jejuni subsp. jejuni, Camp. coli (two clusters) and Camp. lari. The study shows that strains within each species are diverse genomically. Both molecular methods were highly discriminatory, although some strains with identical ribotypes could be distinguished by AP-PCR, and they are valuable new alternatives to traditional typing in epidemiological studies of environmental campylobacters.

Spontaneous Transfer of the Ti Plasmid ofAgrobacterium tumefaciensand the Nopaline Catabolism Plasmid ofA. radiobacterStrain K84 in Crown Gall Tissue

Spontaneous transfer of the Ti plasmid from Agrobacterium tumefaciens to strain K84 of A. radiobacter was observed and studied for the first time in an experiment on biological control of crown gall. This transfer was detected in a tumor from a K84-treated plant grown in soil inoculated with a nopaline strain of A. tumefaciens biovar 1 sensitive to agrocin 84. The transconjugant strain was virulent and produced agrocin 84. Southern blot hybridization analysis with several probes (T-DNA and right adjacent regions and vir genes) showed important changes at the Ti plasmid, suggesting that recombination between Ti plasmid and pAtK84b in K84 could have happened, resulting in a new Ti plasmid. Transfer of both plasmids of strain K84, pAtK84b and pAgK84, responsible for nopaline catabolism and agrocin 84 production, respectively, to A. tumefaciens also was detected in isolates from the same tumor. Southern blot hybridization of plasmids from one of these avirulent isolates with a nopaline plasmid-specific probe of strain K84 indicated there was a replacement of Ti plasmid by pAtK84b in A. tumefaciens, explaining its avirulence. These results show that plasmid exchanges can occur spontaneously between A. tumefaciens and A. radiobacter. This kind of transfer generates genetic diversity in Agrobacterium and may influence the biocontrol efficiency of A. radiobacter.

Factors affecting Agrobacterium tumefaciens-mediated transformation in several black poplar clones

Transient expression of the uidA reporter gene was used in preliminary experiments with two oncogenic and two disarmed Agrobacterium tumefaciens strains in order to test the efficiency of T-DNA transfer to N084 x Populus nigra and N107 x P. nigra clones. The oncogenic strain A281 pKIWI105 produced the highest average number of GUS spots per leaf disc. In order to optimize the production of transgenic plantlets from different P. nigra clones (San Giorgio, Jean Pourtet, N084 x P. nigra and N107 x P. nigra, respectively), two A. tumefaciens strains (GV2260 p35S GUS, A281pKIWI105) and bacterial concentrations (7×108; 1.2×09 bacteria ml-1) were used. Following co-cultivation with A281 pKIWI105, the frequency of leaf discs producing kanamycin-resistant calli was not significantly different between the clones and bacteria concentrations used. Transformed shoots were regenerated from all clones, except for Jean Pourtet. Co-cultivation of leaf discs with GV2260 p35S GUS produced very few calli which died when transferred to selective regeneration medium. In addition, the effects of acetosyringone and leaf wounding were evaluated for the San Giorgio and Jean Pourtet clones, using the same strains. Factors which significantly affected the transformation efficiency of leaf explants were the P. nigra clone, the A. tumefaciens strain, and the presence of acetosyringone. Genetic transformation of calli and regenerated plantlets was confirmed by their ability to grow and root on Woody Plant Medium containing kanamycin, by histochemical β-glucuronidase assays, and Southern blot hybridization analyses.

The germ cell deficient locus maps to mouse Chromosome 11A2?3

The autosomal recessive mouse mutation, germ cell deficient, gcd, manifests as infertility in both sexes owing to improper migration and/or proliferation of primordial germ cells during embryonic development. Mice harboring this mutation have been hypothesized to be animal models of the human syndromes, premature ovarian failure and Sertoli cell only syndrome. Since the gcd mutation arose from the insertion of over 100 kb of foreign DNA into the chromosome during a transgenic mouse experiment, fluorescent in situ hybridization with the transgene as a probe was used to determine the chromosomal position of the gcd locus. DAPI chromosomal banding in conjunction with double labeling with the alpha 1(I) collagen gene revealed that the gcd locus is situated on mouse Chromosome (Chr) 11A2-3. Two candidate genes, Lif and Oncostatin M, map near the gcd locus; however, Southern blot hybridization analysis revealed no gross rearrangements in these genes in gcd mice. The chromosomal position of the gcd locus will prove valuable in the search for other candidate genes as well as a landmark for positional cloning experiments.

Transovarian transmission of a foreign gene in the silkworm, Bombyx mori, by Autographa californica nuclear polyhedrosis virus.

We introduced a firefly luciferase gene, expressed under control of Drosophila heat shock protein gene promoter, into Autographa californica nuclear polyhedrosis virus (AcNPV). When the 5th instar larvae of the silkworm, Bombyx mori, were inoculated with the recombinant virus, luciferase activities were detected in the virus-infected larvae and pupae, and in the newly hatched larvae of the next generation. PCR amplification and Southern blot hybridization analysis demonstrated that the luciferase gene was transmitted through at least the F2 generation. In addition, the V-cathepsin gene, encoding a cysteine protease of AcNPV, was also detected in the DNA of all individuals of the F2 generation. These results show that AcNPV can be utilized as vector for the transovarian transmission of foreign genes in the silkworm.

Horizontal transfer of a mitochondrial plasmid.

Direct evidence for horizontal transfer of a mitochondrial plasmid from the discomycete Ascobolus immersus to the pyrenomycete Podospora anserina is presented. Southern blot hybridisation analysis, polymerase chain reaction (PCR) amplification, and DNA sequencing demonstrate transmission of a linear plasmid upon hyphal contact. DNA extraction from isolated organelles indicates a mitochondrial localisation for the plasmid in P. anserina. This is the first report of horizontal gene transfer among unrelated fungi. These results have important evolutionary implications for plasmid propagation in fungi.

Enhanced Expression of ICAM-1 in a Murine Fibrosarcoma Reduces Tumor Growth Rate

Intercellular adhesion molecule-1 (ICAM-1) plays an essential role in lymphocyte adhesion to endothelium and migration across endothelial cell barriers. We undertook this study to determine the growth of a murine fibresarcoma transfected with the ICAM-1 gene. MCA-105 tumor cells were cotransfected with ICAM-1 and the plasmid for neomycin resistance (NeoR). Selected G418-resistant clones were expanded and cell surface ICAM-1 expression was verified using a fluorescence-activated cell sorter. Integration of the ICAM-1 gene and ICAM-1 mRNA expression were verified by Southern and Northern blot hybridization analysis, respectively. C57BL/6 mice were divided into five groups (six animals/group): Control, NeoR only, ICAM-1 (low expressing, Clone 25), ICAM-1 (high expressing, Clone 81), and a 1:1 mixture of NeoR:Clone 81; animals received 1 × 10 6 cells on Day 0 and tumor measurements began on Day 7 and were measured in mm 2. At 19 days, tumors from cell lines expressing ICAM-1 were significantly ( P < .05) smaller than both the parental cell line and tumor-containing NeoR only (364 mm 2 vs 466 mm 2 and 527 mm 2, respectively). This decrease in tumor growth may be a result of increased lymphocyte migration or increased anti-tumor cytotoxicity by infiltrating lymphocytes. The results from the mixed tumor experiment suggest a possible paracrine effect by cells expressing ICAM-1. Studies are currently under way to investigate the effect of immunotherapy on tumors derived from ICAM-1-cloned transfectants.

Leptospira interrogans serovar sejroe infection in a group of laboratory dogs.

Interstitial nephritis was seen histologically in 19 (59%) out of 32 pure-breed beagle dogs (16 males and 16 females) subjected to standard safety tests. In these animals no clinical abnormalities were observed and all the tested parameters (haematology, biochemistry and urine analysis) were within the normal ranges. Leptospiral antibody titres ranging from 1 : 100 to 1 : 6400, against a serovar (hardjo) belonging to the Sejroe serogroup, were detected by the microscopic agglutination test (MAT) in the serum of the 19 dogs with interstitial nephritis. All animals without renal lesions were seronegative. Leptospiral antigen was detected immunohistochemically in the kidneys of 4 dogs; leptospires were detected in Warthin-Starry stained sections of one dog. Leptospires were isolated from the kidneys of 3 of the 4 dogs examined by bacterial culture. The isolated strains were typed as serovar sejroe by restriction endonuclease digestion and Southern blot hybridization analysis of their DNA. It was concluded that Leptospira interrogans serovar sejroe, was responsible for an asymptomatic chronic renal infection which was widespread in this group of laboratory dogs.

Agrobacterium-mediated transformation and regeneration of fertile transgenic plants of chinese cabbage (brassica campestris ssp. pekinensis cv. ‘spring flavor’)

A procedure for the regeneration of fertile transgenic Chinese cabbage (Brassica campestris ssp. pekinensis cv. 'Spring Flavor') is presented in this report. The protocol is based on infection of cotyledon explants of 5-d-old seedlings with an Agrobacterium tumefaciens strain LBA4404 carrying a disarmed binary vector pTOK/BKS-1. The T-DNA region of this binary vector contains the nopaline synthase/neomycin phosphotransferase II (nptII) chimeric gene for kanamycin resistance and the cauliflower mosaic virus 35S/coat protein gene of tobacco mosaic virus L (TMV-L) chimeric gene. After co-cultivation for 48 h, the cotyledonary petioles were placed on shoot induction media containing 15 mg/L kanamycin sulfate. Shoot induction was continued for 3-4 weeks, then subcultured once and after 2 weeks the shoots were transferred to root induction medium. After 1 week 8 putatively transformed plantlets from 200 cotyledon explants were obtained and transferred to greenhouse. Six of them grew to maturity, produced normal flowers and set seeds. Polymerase chain reaction and Southern blot hybridization analyses confirmed the introduction of the T-DNA into the Chinese cabbage genome. Further, Western blot analysis using polyclonal TMV antiserum showed most of the regenerants (5 out of 6) expressed TMV coat protein gene. Stable inheritance of the inserted clone was investigated in the next generation.

Association of human papillomavirus with malignant and premalignant lesions of the uterine endometrium

The possible association of human papillomavirus (HPV) with endometrial hyperplasia and endometrial adenocarcinoma was investigated. DNA from frozen tissues of 30 endometrioid carcinomas of Japanese patients was tested for HPV DNA by Southern blot hybridization analysis. Screening with HPV type 58 probe under low stringency conditions showed the presence of HPV DNA in two of 30 endometrioid carcinomas. High stringency hybridization identified HPV type 16 in the two positive specimens. The presence of HPV was further analyzed by polymerase chain reaction (PCR)-Southern blot analysis of DNA from archival tissue blocks of the initial 30 endometrioid carcinomas as well as an additional 17 endometrioid carcinomas and 13 atypical hyperplasias of the endometrium from Japan and 38 endometrioid carcinomas from the United States. Polymerase chain reaction amplification using type 16-specific HPV primers for a portion of the E6 open reading frame was positive in six of 47 (13%) endometrioid carcinomas from Japan, including two in which HPV 16 was not detected by Southern blot analysis and two of 38 (5%) endometrioid carcinomas from the United States. Polymerase chain reaction amplification using Ll consensus sequence primers was positive for HPV in two of 13 (15%) endometrial hyperplasias, 13 of 47 (28%) endometrioid carcinomas from Japan, and six of 38 (16%) endometrioid carcinomas from the United States. Slot blot hybridization identified HPV type 16 in seven of the L1 PCR products, including all but one specimen testing positive for HPV type, 16 using E6 type specific primers. In situ hybridization was positive for HPVs 16/18 in glandular epithelial tumor cells in six of the PCRpositive specimens. An additional specimen showed staining for HPVs 16/18 in acellular luminal debris in association with squamous metaplasia of the tumor, but staining was negative in the glandular cells of the tumor. Human papillomavirus was not detected by in situ hybridization in the remaining specimen, which was PCR positive for HPV 16. In situ hybridization was weakly positive for HPVs 31/33/ 35 in one specimen and was weakly positive for HPVs 6/11 in benign endometrial epithelial cells but not in tumor cells of another specimen that tested positive for HPV by Ll PCR. Two dimensional gel electrophoresis performed on two specimens showed that HPV DNAs were integrated into cellular DNA with no episomal coexistence. These findings suggest that HPV, especially HPV 16, may play an etiologic role in a fraction of endometrioid adenocarcinomas.

Characterization of the pTFI91-family replicon of Thiobacillus ferrooxidans plasmids.

Plasmids found in six strains of Thiobacillus ferrooxidans were mapped and compared in an effort to detect the origin of replication. Four strains yielded an identical 9.8-kb plasmid, pTFI91. Restriction mapping and Southern blot hybridization analysis were used to confirm this finding. Dissimilar plasmids found in two other strains contained a conserved 2.2-kb SacI region common to pTFI91. DNA sequence analysis of this region showed structural features common to bacterial plasmid replicons. A comparison of the pTFI91 origin with those of T. ferrooxidans pTF-FC2 and other broad host range vectors did not show significant homologous DNA sequences. To verify the replication function, a chloramphenicol acetyl transferase marker gene was ligated at the unique sites of pTFI91, and the plasmid was transformed into Escherichia coli DH5 alpha cells but no transformants were identified. To test the replication of pTFI91 independent of DNA polymerase I in E. coli, different restriction fragments of pTFI91 were cloned into pHSG398 (Cmr, ColEI origin) and transformed into the polA1 mutant SF800, but chloramphenicol-resistant transformants were not detected. Electrotransformation of T. ferrooxidans TFI-70 and Pseudomonas putida ATCC 19151 also failed to yield transformants. The results suggested that the pTFI91 plasmid replicon does not function either in E. coli or in P. putida. Since pTFI91 contains the same origin of replication as other plasmids in several other T. ferrooxidans strains, this replicon may be commonly distributed in T. ferrooxidans.