Cosmid DNA Research Articles

Rapid isolation of genes from an indexed genomic library of C. cinereus in a novel pab1 + cosmid

In this study we present an indexed genomic library of homokaryon AmutBmut constructed within a novel cosmid carrying pab1 + as a selectable Coprinus marker. The average insert size per cosmid comprises 41 kb. We screened the library and detected copies of known ( a1–2, β-tub, cgl1, ras, trp1) and of new Coprinus genes ( cac, lac1, lac2, lac3). Screening was performed either by Southern blot hybridisation or more efficiently by non-radioactive PCR amplification. We successfully applied PCR with specific and with degenerate primers, multiplex PCR and colony PCR in library screening. Our results suggest a new, more efficient pooling strategy for future high throughput screenings to be used in PCR with pooled cosmid DNAs, or in a less laborious approach using pooled Escherichia coli colonies for PCR.

Gene Delivery Using Helper Virus–Free HSV‐1 Amplicon Vectors

AbstractHerpes simplex virus type 1 (HSV‐1)‐based amplicon vectors contain only a very small percentage of the 152‐kbp viral genome. Consequently, replication and packaging of amplicons depend on helper functions that are provided either by replication‐defective mutants of HSV‐1 or by replication‐competent, but packaging‐defective, HSV‐1 genomes. Sets of cosmids that overlap and represent the entire HSV‐1 genome can form, via homologous recombination, circular replication‐competent viral genomes, which give rise to infectious virus progeny. However, if the DNA cleavage/packaging signals are deleted, reconstituted virus genomes are not packageable, but still provide all the helper functions required for the packaging of cotransfected amplicon DNA. The resulting stocks of packaged amplicon vectors are essentially free of contaminating helper virus. This unit describes the cotransfection of amplicon and cosmid DNA into 2‐2 cells by cationic liposome‐mediated transfection using and the harvesting of packaged vector particles. Support protocols provide methods for preparing cosmid DNA and determining the titers of amplicon stocks.

The murine p75 TNF receptor promoter region: DNA sequence and characterization of a cis-acting silencer

The promoter region of the murine p75 TNF receptor (TNF-R) was isolated from a mouse genomic DNA cosmid library. The promoter region is devoid of TATA box and has the characteristics of a house keeping gene since it contains multiple SP1 binding sites. Its mRNA has different inititiation sites in different lymphoid cell lines. This promoter confers transcriptional activity to heterologous reporter genes. Deletion analysis showed that there is a silencer element located upstream from position −841. This inhibitory sequence is active both in fibroblasts and T-cell lines transiently or permanently transfected. A fragment from −929 to −841 is capable of transferring this ‘silencer’ activity to the early SV40 promoter. This activity could be blocked in trans- when a plasmid containing the same sequence was co-transfected with the reporter plasmid indicating that a protein binds to this region of the promoter.

High resolution mapping DNAs by R-loop atomic force microscopy.

R-loops formed by short RNA transcripts have been imaged by atomic force microscopy (AFM) at a constant force in the height mode. The technique was applied to mapping the human endogenous retrovirus K10 family (HERV-K10) long terminal repeats (LTR) within individual plasmids and cosmids. RNA probes specific for the U3 (384 nt) and U5 (375 nt) LTR regions separated by a span of 200 bp were used for R-loop formation with LTRs located within plasmid (3.8 kb) or cosmid ( approximately 40 kb) DNAs. R-loops stabilized by glyoxal treatment and adsorbed onto the mica surface in the presence of magnesium ions looked like looped out segments of RNA:DNA hybrids. The total yield of R-loops was usually approximately 95%. The RNA:DNA hybrids were found to be 12-15% shorter than the corresponding DNA:DNA duplex. The two regions of the LTR could be easily discerned in the AFM images as clearly separated loops. R-loop positions determined on cosmids by AFM were accurate to approximately 0.5% of the cosmid length. This technique might be easily adapted for mapping various sequences such as gene exons or regulatory regions and for detecting insertions, deletions and rearrangements that cause human genetic diseases.

Genomic identification of the minor histocompatibility antigen HA-1 locus by allele-specific PCR.

Graft-versus-host disease (GvHD) can be a major complication of allogeneic bone marrow transplantation even in recipients of HLA genotype-identical transplants. Disparities in minor histocompatibility antigens (mHags) between donor and recipient are a potential risk for the development of GvHD. A mismatch for the mHag HA-1 can cause GvHD in adult recipients of allogeneic bone marrow from HLA-identical donors. The mHag HA-1, first identified by HLA-A*0201-restricted cytotoxic T cells (CTLs), was recently chemically characterized as a nonapeptide. On the cDNA level, the HA-1 locus has two alleles, HA-1H and HA-1R, which differ in two nucleotides, resulting in a single amino acid substitution. Here we report on the genomic structure of the HA-1 locus. Isolation and sequencing of cosmid DNA encoding the HA-1 peptide sequence revealed that the HA-1 alleles are encoded by two exons. Two different primer sets were designed, each consisting of allele-specific primers and a common primer, and both sets containing intronic sequences. We performed genomic DNA typing of three families consisting of 24 HLA-A*0201-positive individuals. The predicted allele-specific products correlated in all cases with the mHag classification by CTLs and by RT-PCR. We demonstrate for the first time the genomic identification of the mHag HA-1 locus. Prospective genomic typing for the HA-1 alleles will improve donor selection and identify HLA-A*0201-positive recipients with a high risk for HA-1-induced GvHD.

Importance of cytogenetic markers for multiple primary carcinomas in colorectal cancer: chromosome 17 and p53 locus translocation.

The incidence of non-familial multiple primary cancer in colorectal cancer patients has increased in recent years in Japan. To clarify the characteristic genetic aberrations in such multiple cancers, we examined structural chromosomal aberrations by fluorescence in situ hybridization, using chromosome 17-specific and p53 cosmid DNA probes. We established short-term cultures of 78 surgical specimens and were able to obtain observable metaphase spreads in 23 single colorectal cancer specimens and in 6 colorectal cancer specimens from patients with double primary cancers. The frequency of chromosome 17 and/or p53 locus translocation was significantly greater in tumors with double cancer than in single colorectal cancers (P < 0.05 and P < 0.01, respectively). These aberrations in double cancers frequently appeared even at an early Dukes' stage (A and B) of colorectal carcinoma. Our results suggest that translocation of chromosome 17 and the p53 locus may be specific genetic events probably associated with carcinogenesis of multiple primary cancers in colorectal cancer.

Characterization of an acidic-pH-inducible stress protein (hsp70), a putative sulfatide binding adhesin, from Helicobacter pylori.

The in vitro glycolipid binding specificity of the gastric pathogen Helicobacter pylori is altered to include sulfated glycolipids (sulfatides) following brief exposure of the organism to acid pH typical of the stomach. This change is prevented by anti-hsp70 antibodies, suggesting that hsp70 may be a stress-induced surface adhesin, mediating sulfatide recognition. To facilitate investigation of the role of hsp70 in attachment, we have cloned and sequenced the H. pylori hsp70 gene (dnaK). The hsp70 gene was identified by probing a cosmid DNA library made from H. pylori 439 with a PCR amplicon generated with oligonucleotides synthesized to highly conserved regions of dnaK. The 1.9-kb H. pylori hsp70 gene encodes a product of 616 amino acids. Primer extension analysis revealed a single transcription start site, while Northern blot analysis established that hsp70 was preferentially induced by low pH rather than by heat shock. The ability of H. pylori to alter its glycolipid binding specificity following exposure to low pH by upregulating hsp70 and by expressing hsp70 on the bacterial surface may provide a survival advantage during periods of high acid stress.

Varicella-Zoster Virus ORF61 Deletion Mutants Replicate in Cell Culture, but a Mutant with Stop Codons in ORF61 Reverts to Wild-Type Virus

Varicella-zoster virus (VZV) ORF61 encodes a phosphoprotein that transactivates VZV promoters. Transfection of cells with cosmid DNAs, including a cosmid with a large deletion in ORF61, resulted in a VZV ORF61 deletion mutant that was impaired for growthin vitroand could be partially complemented by growth in neuroblastoma or osteosarcoma cell lines. Cells infected with the VZV ORF61 deletion mutant expressed normal levels of an immediate-early VZV protein, but had reduced levels of a late protein and showed abnormal syncytia. Carboxy terminal truncation mutants of VZV ORF61 protein have a transrepressing phenotype and inhibit the infectivity of cotransfected wild-type viral DNA. Transfection of cells with cosmid DNAs, including a cosmid with stop codons that should result in an ORF61 truncation mutant expressing a transrepressing protein that retains the RING finger domain, resulted in a viral genome which reverted back to the wild-type sequence. BAL-31 exonuclease was used to produce deletions at the site of the stop codons in ORF61 of the cosmid, resulting in loss of the RING finger domain. Transfection of tissue culture cells with the ORF61 BAL-31 deletion mutants and other cosmid DNAs yielded viable viruses. Thus, while deletion mutants lacking the RING finger domain of ORF61 replicate in cell culture, a mutant with stop codons that retains this domain could not be propagated and reverted to wild-type virus.

SOLH, a Human Homologue of theDrosophila melanogaster small optic lobesGene Is a Member of the Calpain and Zinc-Finger Gene Families and Maps to Human Chromosome 16p13.3 nearCATM(Cataract with Microphthalmia)

Mutations in theDrosophila melanogaster small optic lobes(sol) gene cause a severe reduction in the neuropiles of the medulla and lobula complexes of the adult optic lobes. The predicted protein product ofsolcontains zinc-finger-like repeats, a calpain-like protease domain, and a C-terminal region of unknown function. We have isolated human brain cDNA forSOLH, a human homologue ofsol. The humanSOLHgene consists of 14 exons distributed over more than 45 kb of genomic DNA. The encoded SOLH protein of 1086 amino acids has strong similarity to theD. melanogasterprotein. The calpain-like domain and C-terminal region are highly conserved (58% identity), and similar Cys2-Cys2zinc fingers are present in the N-terminal region. A reportedCaenorhabditis eleganshomologue contains the calpain domain and C-terminal region, but appears to lack the zinc finger region. A single copy of the zinc finger sequence is present in adjacentC. elegansgenomic cosmid DNA sequence, and we show that it is part of theC. elegans sol-like transcript. Northern analysis of human tissues revealed aSOLHtranscript of ∼5 kb that was strongest in human brain. We have mapped theSOLHgene to chromosome 16p13.3 byin situhybridization.SOLHis a candidate gene forCATM(hereditary cataracts with microphthalmia), which maps in this region.

Identification of an additional member of the secretin superfamily of proteins in Pseudomonas aeruginosa that is able to function in type II protein secretion.

Pseudomonas aeruginosa is a prolific exporter of virulence factors and contains three of the four protein secretion systems that have been described in gram-negative bacteria. The P. aeruginosa type II general secretory pathway (GSP) is used to export the largest number of proteins from this organism, including lipase, phospholipase C, alkaline phosphatase, exotoxin A, elastase and LasA. Although these exoproteins contain no sequence similarity, they are specifically and efficiently transported by the secretion apparatus. Bacterial homologues of XcpQ (GspD), the only outer membrane component of this system, have been proposed to play the role of gatekeeper, by presumably interacting and recognizing the exported substrates to allow their passage through the outer membrane. While determining the phenotype of nonpolar deletions in each of the xcp genes, we have shown that a deletion of the P. aeruginosa strain K xcpQ does not completely abolish protein secretion. As the proposed function of XcpQ should be requisite for secretion, we searched for additional factors that could carry out this role. A cosmid DNA library from a PAK strain deleted for xcpP-Z was tested for its ability to increase protein secretion by screening for enhanced growth on lipid agar, a medium that selects for the secretion of lipase. In this manner, we have identified an XcpQ homologue, XqhA, that is solely responsible for the residual export observed in a deltaxcpQ strain, although it is not required for efficient secretion in wild-type P. aeruginosa. We have also demonstrated that this protein is capable of recognizing all of the exoproteins of P. aeruginosa, arguing against the proposed role of members of the secretin family as determinants of specificity.

Cloning and chromosomal localization of a paralog and a mouse homolog of the human transaldolase gene

A sequence homologous to the transaldolase gene ( TALDO) was identified in a polymorphic cosmid DNA mapped on human chromosome 11p15 by exon trapping with pSPL3. Analysis of lambda clones contiguous to the cosmid clone showed that the related gene ( TALDOR) consists of 8 exons spanning approximately 19 kb from the translation start site to the polyadenylation signal. The exon sequence of TALDOR was almost identical with that of TALDO localized on 1p33-34.1, but its exons corresponding to exons 4 and 5 of TALDO were found to be split by 4 introns in TALDOR. To examine the evolutionary conservation of two genes for transaldolase, we have isolated the cDNA for its mouse homolog and determined the nucleotide sequence covering the complete coding region. Fluorescence in situ hybridization using the cDNA as a probe showed that the mouse transaldolase gene ( Taldo) is localized on chromosome 7 F3-F4 as a single copy gene. This chromosomal region is known to be syntenic to human chromosome 11p15 rather than to 1p33-p34.1, suggesting that TALDOR is the ancestral form. The existence of TALDOR implies a duplication of the mammalian transaldolase gene after divergence of rodent and primate.

A New Complex Translocation (15;20;17)(q22;p13;q21) in Acute Promyelocytic Leukemia

We describe here a 39-year-old male with acute promyelocytic leukemia (APL) carrying a new complex translocation (15;20;17). A chromosomal analysis of the bone marrow cells showed 46,XY, t(15;20;17)(q22;p13;q21). Fluorescence in situ hybridization (FISH) analysis using plasmid DNA libraries of chromosomes 15, 17, and 20 revealed three derivative chromosomes, der(15)t(15;17), der(17)t(17;20), and der(20)t(15;20). Fluorescence in situ hybridization with cosmid DNA probes flanking the breakpoints of t(15;17) did not show the retinoic acid receptor α (RARα)/PML fusion signal usually generated on the der(17)t(15;17). However, rearrangement of the RARα gene and expression of the PML/RARα chimeric transcript were identified by Southern blot and reverse-transcriptase polymerase chain reaction (RT–PCR) analyses, respectively. Our results confirmed that the PML/RARα gene on the der(15)t(15;17), not the RARα/PML gene, must be essential to leukemogenesis in APL. Furthermore, considering another reported case with a 20p13 aberration, it is possible that 20p13 is a nonrandom breakpoint in APL with a complex translocation.

Reliable and accurate sequencing of lambda, cosmid and P1 DNAs using modified dye terminator reaction parameters

▼Many laboratories still find the direct sequencing of large DNA templates (i.e. lambda, cosmid and bacteriophage P1 clones) problematic. To date, only limited amounts of sequence information, which in many cases is of poor quality, can be generated from such molecules, even when using polymerase chain reaction (PCR)-based cycle sequencing protocols (Ref. 1, 3, 4, 5, 6, 7). To obtain reliable nucleotide data, it is usually necessary to subclone portions of the DNA of interest into either a high copy-number plasmid or a single-strand bacteriophage, such as M13. It would be advantageous to have a simple and rapid protocol that could reliably yield large amounts of accurate sequence information and that could be employed in conjunction with an automated fluorescence-based DNA-sequencing apparatus. Here, we describe optimized reaction parameters (template concentration, primer length and cycling conditions) for use with a commercially available dye terminator DNA sequencing kit, and show the excellent quality of the data that can be obtained from either lambda, cosmid or bacteriophage P1 DNAs. This procedure is performed in a single tube, using unmodified oligonucleotide primers, on templates that do not require extensive purification.

UDPGT cDNA expression and UDPGT1 in human liver.

Following expression of UDPGTh1 and UDPGTh2 in Cos-1 cells, each isoform metabolized three types of dihydroxy- or trihydroxy-substituted ring structures, including the 3,4-catechol estrogen (4-hydroxyestrone), estriol and 17-epiestriol, and hyodeoxycholic acid (HDCA), but the UDPGTh2 isozyme was 100-fold more efficient than UDPGTh1. UDPGTh1 and UDPGTh2 are 86% identical overall (76 differences out of 528 amino acids), including 55 differences in the first 300 amino acids of the amino terminus, a domain which confers isoform substrate specificity. The data indicate a high level of conservation in the amino terminus is not required for the preservation of substrate specificity. Analysis of glucuronidation activity encoded by UDPGTh1/UDPGTh2 chimeric cDNAs constructed at their common restriction sites, Sac I (codon 279), Nco I (codon 385), and Hha I (codon 469), showed that nine amino acids between residues 385 and 469 are important for catalytic efficiency, suggesting that this region represents a domain which is critical for catalysis but distinct from that responsible for aglycon selection. Screening of leukocyte DNA cosmid library with human UDPGT-Br1 (1-470 bps) or UDPGT-Br2 (1-450 bps) resulted in three overlapping clones, which were isolated and mapped by endonucleases. Construction of subclones and DNA sequencing, Southern blot analysis revealed that a cluster of 4 exons (132, 88, 220, 1032 bps in one clone) encodes the entire region of 3' identity shared between human UDPGT-phenol, human UDPGT-Br1 and human UDPGT-Br2. A similar strategy but using probes which correspond to the unique regions of human UDPGT-Br1 and human UDPGT-Br2 showed that the exon 1 of UGT1A and UGT1D encodes the unique region of human UDPGT-Br1, and human UDPGT-Br2 and is located 5.6 and 49 Kb, respectively, upstream of the 4 common exons.

Toxoflavin is an Essential Factor for Virulence of Burkholderia glumae Causing Rice Seedling Rot Disease.

Mutants of Burkholderia glumae, the causal agent of rice seedling rot and grain rot diseases, were isolated by transposon (Tn) mutagenesis with Tn4431 harboring a tetracycline (Tc) resistance gene, which was carried on the suicide vector pUCD623. The resulting Tc-resistant colonies were screened for a loss of toxin production as well as for a lack of pathogenicity to rice seedlings. One of the screened mutants, the mutant No.19, was shown to have a single copy of Tn insertion on the genomic DNA by Southern hybridization test. Restriction enzyme fragments containing Tn4431-flanking sequences from the genomic DNA of mutant No.19 were cloned and used as probes for colony hybridization of the genomic DNA cosmid library from a wild strain of B. glumae. Six recombinant clones showing homology to the probes were isolated and conjugated into mutant No.19 by triparental mating. The recombinant cosmid pNP147, one of six clones, was able to complement the mutant No.19 for toxin production and pathogenicity. The toxin, purified from a culture of mutant No.19 with pNP147, was identified as toxoflavin by NMR and Mass spectra analyses. These results suggest that toxoflavin is a critical factor in disease development of B. glumae, since the nontoxigenic mutants were unable to induce seedling rot disease to rice plants.

Streptomyces coelicolor DNA homologous with acyltransferase domains of type I polyketide synthase gene complex

An acyltransferase-homologous DNA fragment was amplified in a PCR reaction on a cosmid DNA template from the genomic DNA library of the soil bacterium Streptomyces coelicolor A3(2). The putative amino acid sequence of the fragment resembles acyl-CoA:ACP acyltransferase domains from several bacterial enzymatic complexes of polyketide synthase. There is a high similarity with acyltransferase domains from so-called type I polyketide synthases. Such synthases catalyze production of the aglycone portion of macrolides and polyethers that are important as antibiotics or immunosuppressants. The amplified fragment is considered to be a part of a larger gene complex.

A 37-kb restriction map of the human immunoglobulin lambda variable locus, VB cluster, harboring four functional genes and two non-coding V&lt;FONT FACE=Symbol&gt;l&lt;/font&gt; sequences

The human immunoglobulin lambda variable locus (IGLV) is mapped at chromosome 22 band q11.1-q11.2. The 30 functional germline v-lambda genes sequenced untill now have been subgrouped into 10 families (V&lt;FONT FACE="Symbol"&gt;l&lt;/font&gt;1 to V&lt;FONT FACE="Symbol"&gt;l&lt;/font&gt;10). The number of V&lt;FONT FACE="Symbol"&gt;l&lt;/font&gt; genes has been estimated at approximately 70. This locus is formed by three gene clusters (VA, VB and VC) that encompass the variable coding genes (V) responsible for the synthesis of lambda-type Ig light chains, and the J&lt;FONT FACE="Symbol"&gt;l&lt;/font&gt;-C&lt;FONT FACE="Symbol"&gt;l&lt;/font&gt; cluster with the joining segments and the constant genes. Recently the entire variable lambda gene locus was mapped by contig methodology and its one- megabase DNA totally sequenced. All the known functional V-lambda genes and pseudogenes were located. We screened a human genomic DNA cosmid library and isolated a clone with an insert of 37 kb (cosmid 8.3) encompassing four functional genes (IGLV7S1, IGLV1S1, IGLV1S2 and IGLV5a), a pseudogene (V&lt;FONT FACE="Symbol"&gt;l&lt;/font&gt;A) and a vestigial sequence (vg1) to study in detail the positions of the restriction sites surrounding the V&lt;FONT FACE="Symbol"&gt;l&lt;/font&gt; genes. We generated a high resolution restriction map, locating 31 restriction sites in 37 kb of the VB cluster, a region rich in functional V&lt;FONT FACE="Symbol"&gt;l&lt;/font&gt; genes. This mapping information opens the perspective for further RFLP studies and sequencing

Cloning and expression of the porcine obese gene

The product of the obese gene, leptin, may be an important regulator of adiposity via its regulation of feed intake and energy metabolism. Probes were developed using the polymerase chain reaction to analyze gene expression and determine the structure of the porcine ob gene. Porcine ob was expressed in adipose tissue as a 3,100 bp mRNA. Finished pigs (136 kg) had higher (P<.01) levels of ob mRNA (per unit of 6‐actin mRNA) in subcutaneous adipose tissue than did growing pigs (60 kg). Obese gene expression was not detected in tissues other than adipose depots. A genomic DNA fragment containing the ob gene was isolated from a cosmid DNA library. Sequence analysis indicates that the ob gene has three exons. A short untranslated sequence was identified as exon 1 and the amino acid coding sequence was located in the second and third exons. The gene structure, intron/exon boundaries, and the amino acid sequence was highly conserved in mammalian species. The porcine leptin amino acid sequence was 95%, 92% and 89% similar to cattle, human and mouse leptin sequences, respectively.

Direct primer walking on P1 plasmid DNA.

BioTechniquesVol. 23, No. 1 BenchmarksOpen AccessDirect Primer Walking on P1 Plasmid DNAV. Benes, C. Kilger, H. Voss, S. Pääbo & W. AnsorgeV. BenesEuropean Molecular Biology Laboratory, HeidelbergSearch for more papers by this author, C. Kilger*Address correspondence to Christian Kilger, Institute of Zoology, University of Munich, P.O. Box 202126, D-80021 Munich, Germany. Internet: E-mail Address: kilger@zi.biologie.uni-muenchen.deEuropean Molecular Biology Laboratory, HeidelbergSearch for more papers by this author, H. VossEuropean Molecular Biology Laboratory, HeidelbergSearch for more papers by this author, S. PääboEuropean Molecular Biology Laboratory, HeidelbergSearch for more papers by this author & W. AnsorgeEuropean Molecular Biology Laboratory, HeidelbergSearch for more papers by this authorPublished Online:7 Aug 2018https://doi.org/10.2144/97231bm21AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail FiguresReferencesRelatedDetailsCited ByUnlocking the mystery of the hard-to-sequence phage genome: PaP1 methylome and bacterial immunity19 September 2014 | BMC Genomics, Vol. 15, No. 1Exploring the utility of an indel-rich, mitochondrial intergenic region as a molecular barcode for bamboo corals (Octocorallia: Isididae)Marine Genomics, Vol. 2, No. 3-4Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles10 March 2003 | Nature Biotechnology, Vol. 21, No. 4Comparative genomic analysis in the region of a major Plasmodium -refractoriness locus of Anophelesgambiae11 June 2002 | Proceedings of the National Academy of Sciences, Vol. 99, No. 12From First Base: The Sequence of the Tip of the X Chromosome of Drosophila melanogaster , a Comparison of Two Sequencing Strategies1 May 2001 | Genome Research, Vol. 11, No. 5BAC Trimming: Minimizing Clone OverlapsGenomics, Vol. 64, No. 1Gene Walking by PCR Amplification of Short Fragments from Taq DNA Polymerase—Modified P1 Plasmid DNA and TA CloningBéatrice Baury, Damien Masson, Patrick Lustenberger & Marc G. Denis22 August 2018 | BioTechniques, Vol. 27, No. 6Cloning and Characterization of the Murine Histone Deacetylase (HDAC3)Biochemical and Biophysical Research Communications, Vol. 263, No. 2Genomic Organization and Chromosomal Localization of the Human Histone Deacetylase 3 GeneGenomics, Vol. 56, No. 2Direct sequencing of long polymerase chain reaction fragmentsMolecular Biotechnology, Vol. 10, No. 2Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophyNature Genetics, Vol. 20, No. 1Chapter 14 Zebrafish YAC, BAC, and PAC Genomic LibrariesReliable and accurate sequencing of lambda, cosmid and P1 DNAs using modified dye terminator reaction parametersTechnical Tips Online, Vol. 3, No. 1 Vol. 23, No. 1 Follow us on social media for the latest updates Metrics Downloaded 166 times History Published online 7 August 2018 Published in print July 1997 Information© 2018 Author(s)PDF download

The role of flagella and motility in the adherence and invasion to fish cell lines by Aeromonas hydrophila serogroup O:34 strains

We compared the ability of Aeromonas hydrophila wild-type strains of serogroup O:34, non-motile Tn 5 aflagellar mutants and the same mutants harboring a recombinant cosmid DNA from a library of A. hydrophila AH-3 (O:34, wild-type) that allows these mutants to make flagella and to be motile, to adhere and invade two fish cell lines. We found that motility is essential in these strains for adhesion, and also that possession of flagella is essential for the ability to invade the fish cell lines. We cannot rule out that flagella may be an adhesin, or that motility may also be involved in A. hydrophila serogroup O:34 bacterial invasion of both fish cell lines.