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PubMed1986

Organization of the human genes for insulin-like growth factors I and II.

Recently, we have reported the isolation of cDNAs encoding the precursors of insulin-like growth factors I and II (IGF-I and II) [(1983) Nature 306, 609-611; (1985) FEBS Lett. 179, 243-246. These cDNAs were employed as specific probes to detect and isolate the corresponding genes from human cosmid DNA libraries. Three cosmids were detected, together containing the entire cDNA sequence of IGF-I, and one cosmid containing the sequence of IGF-II cDNA. Southern blot hybridization, physical mapping and nucleotide sequence analysis of these cosmids revealed that the IGF-I and -II genes have a discontinous structure. The IGF-I gene contains at least four exons spanning a region of probably more that 45 kilobasepairs (kb), while the IGF-II gene consists of at least five exons, spanning a region of 16 kb.

Amino Acid Sequence↗
PubMed1987

Transfection of human lymphocytes with cloned Epstein-Barr virus (EBV) DNA.

Human primary cord-blood lymphocytes were transfected, using the DEAE-dextran technique, with a set of seven largely overlapping clones jointly covering the whole M-ABA Epstein-Barr virus (EBV) genome. Three fragments, cosmids cMB-14 and cM301-99 and plasmid pM966-20, were able to stimulate transient cellular DNA synthesis, blastic transformation, and clumps formation, as well as to prolong the life span from a maximum of 2 weeks in control cultures to up to 6 weeks. The fragments stimulating DNA synthesis also expressed this property in mutual combinations or when combined with cosmid cMSal-A or cM302-21. Their use with any other fragments in cotransfection did not result in further DNA synthesis stimulation. Cosmids cM302-23 and cMSal-B suppressed this effect. Cosmid cM301-99 but not cM302-23 induced transient EBNA-1 formation in about 1% of lymphocytes. Lymphocytes transfected with single fragments or their combinations failed to grow into immortalized cell lines. The results suggest that transient expression of viral functions at levels achievable by transfection is not sufficient for cell immortalization.

Cell Transformation, Viral↗
PubMed1985

Identification of murine H-2Db histocompatibility antigens in cells transfected with cloned H-2 genes.

Clones of mouse L-cells transformed with 21 cosmids containing 15 major histocompatibility complex class I genes of C57BL10 (H-2b) sperm cell DNA were analyzed for the expression of their transfected H-2 and Qa/Tla genes. Three cosmids contained a single gene, mapping to the H-2D region. This gene encodes the H-2Db alloantigen: mouse L-cells transfected with cosmids containing this gene reacted with monoclonal antibodies and alloantisera specific for the H-2Db antigen and expressed a 46-kd H-2 heavy chain associated with beta 2-microglobulin in their cell membranes. Furthermore, these transfected cells were stimulators of, and targets for, anti-H-2Db cytotoxic T-lymphocytes. Eighteen cosmids contained 14 different genes mapping to the Qa and Tla regions. L-cells transfected with these genes did not express class I genes reacting with alloantisera or monoclonal antibodies against Qa2, Qa4 or TL differentiation antigens. In particular, the Qa2,3 gene of C57BL10 was not identified.

Animals↗
PubMed1983

Cloning DNA from the replication terminus region of the Bacillus subtilis chromosome.

DNA from the Bacillus subtilis 168 prototroph, SB19, was partially cleaved with MboI and cloned into the BamHI site of the Escherichia coli cosmid vector, pHC79. [3H]thymine-labelled DNA from the replication terminus region of the B. subtilis dna-1 chromosome was used to identify, by hybridization, clones harboring recombinant cosmids carrying inserts from the terminus region. Restriction maps have been constructed for two such cosmids carrying overlapping DNA inserts that cover or extend into four of the previously identified five SalI fragments which are replicated last. The composite map of the cloned region, together with the available data on the replication order of fragments within it, is consistent with its replication being achieved by the unidirectional movement of a fork through it and towards the late replicating 16.2-kb SalI fragment. Most, if not all, of the terminus sequences in at least one of the recombinant cosmids are missing from a viable strain of B. subtilis that carries a deletion in the SP beta-gltA region of the chromosome.

Bacillus subtilis↗
PubMed1984

Physical map of the Bacillus subtilis replication terminus region: its confirmation, extension and genetic orientation.

The library of Bacillus subtilis DNA previously cloned in the cosmid vector pHC79 has been screened for the replication terminus region using a higher level of terminus probe. 24 of 48 recombinant cosmids which gave a positive response had restriction fragment compositions consistent with their inserts originating from or extending into the terminus region for which a 150-kb restriction map has already been constructed (Weiss and Wake, 1983). DNA spanning terC, the site of termination, appears to be missing from the library, although DNA to either side of terC has been cloned. A detailed analysis of four of the newly identified recombinant cosmids has confirmed most of the previous 150-kb map and allowed it to be extended to 180 kb. Physical linkage of the two cosmid inserts that most closely approach terC on each side has been demonstrated. The location of the genetic marker gltA and the orientation of the restriction map relative to the genetic map of the B. subtilis chromosome have also been established.

Bacillus subtilis↗
PubMed1985

Cloning of genes encoding pectolytic enzymes from a genomic library of the phytopathogenic bacterium, Erwinia chrysanthemi.

Erwinia chrysanthemi are phytopathogenic enterobacteria causing soft-rot disease due to pectolytic enzymes degrading plant cell walls. We constructed a genomic library from Sau3A-digested E. chrysanthemi B374 DNA cloned in the BamHI site of the broad-host-range cosmid pMMB33 grown in Escherichia coli. Out of 1500 kanamycin-resistant (KmR) transductants of E. coli, nine pectolytic-enzyme-positive clones were identified. One of these contained the pEW325 cosmid with a 35-kb insert of Erwinia DNA. Cell extracts of E. coli harboring the cosmid pEW325 were fractionated on a polyacrylamide electrofocusing gel; bands with pectolytic activity were found to co-focus with pectolytic enzymes of E. chrysanthemi B374 strain. Cosmid pEW325 encodes three pectolytic enzymes PL10, PL20 and PL130 with isoelectric points of about 9.3, 9.2 and 4.6, respectively. These enzymes are lyases that cleave polygalacturonate by transelimination, and give rise to unsaturated products. A 15-kb HindIII fragment coding for polygalacturonate lyases was subcloned in pBR322, and a physical map of the resulting plasmid pPL01 was constructed. Starting from the pPL01, various endonuclease-generated fragments were subcloned into pBR322. Genes encoding pectate lyases were localized within an 8-kb fragment (pPL04) and then in a 2.7-kb fragment (pPL03). Polygalacturonate lyases are expressed at various levels; they accumulated in the periplasmic space of E. coli host, whereas E. chrysanthemi secreted these enzymes into the culture medium.

Chromosome Mapping↗
PubMed1985

Isolation of a human tissue-type plasminogen-activator genomic DNA clone and its expression in mouse L cells.

We have isolated a cDNA clone corresponding to a substantial portion of the human tissue-type plasminogen activator (t-PA) protein. It encodes almost all of the protein B chain and part of the 3' untranslated region. We have used this clone to screen bacteriophage lambda and cosmid libraries of human genomic DNA. Several related genomic clones were isolated. One of these, a cosmid clone, carried approx. 40 kb of human DNA. Mapping experiments indicate that the region containing the protein-coding exons is approx. 20 kb in length. The cosmid, containing the t-PA gene and the aminoglycosyl-3'-phosphotransferase dominant-selection marker, was introduced into mouse L cells. Approximately half of the transformants were shown to produce human t-PA. We demonstrated that the fibrinolytic t-PA activity could be specifically quenched by anti-t-PA antibody and that the recombinant t-PA was of similar size (by SDS-polyacrylamide gel electrophoresis) to the t-PA produced by the human Bowes melanoma cell line. Our results suggest that the cosmid clone carries the whole t-PA coding region together with the regulatory elements necessary for its expression.

Amino Acid Sequence↗
PubMed1992

Chromosome 16-specific repetitive DNA sequences that map to chromosomal regions known to undergo breakage/rearrangement in leukemia cells.

Human chromosome 16-specific low-abundance repetitive (CH16LAR) DNA sequences have been identified during the course of constructing a physical map of this chromosome. At least three CH16LAR sequences exist and they are interspersed, in small clusters, over four regions that constitute more than 5% of the chromosome. CH16LAR sequences were observed in one unusually large cosmid contig (number 55), where the ordering of clones was difficult because these sequences led to false overlaps between noncontiguous clones. Contig 55 contains 78 clones, or approximately 2% of all the clones contained within the present cosmid contig physical map. Fluorescent in situ hybridization of multiple clones, including cosmid and YAC contig 55 clones, mapped the four CH16LAR-rich regions to bands p13, p12, p11, and q22. These regions are of biological interest since the pericentric inversion and the interhomologue translocation breakpoints commonly found in acute nonlymphocytic leukemia (ANLL) subtype M4 fall within these bands. Sequence analysis of a 2.2-kb HindIII fragment from a cosmid containing a CH16LAR sequence indicated that one of the CH16LAR elements is similar to a minisatellite sequence in that the core repeat is only 40 bp in length. Additional characterization of other repetitive elements is in progress.

Animals↗
PubMed2006

[Cloning and analysis of geldanamycin partial biosynthetic gene cluster of Streptomyces hygroscopicus 17997].

A geldanamycin (GDM) producing strain, Streptomyces hygroscopicus 17997, was isolated from Yunnan China soil by our institute researchers. GDM is an ansamycin antibiotic, which has the ability to bind with Hsp90 (Heat Shock Protein 90) and alter its function. Hsp90 is a chaperone protein involved in the regulation of the cell cycle, cell growth, cell survival, apoptosis, and oncogenesis. So it plays a key role in regulating the physiology of cells exposed to environmental stress and in maintaining the malignant phenotype of tumor cells. As an inhibitor of Hsp90, GDM possesses potent antitumor and antivirus bioactivity, but the hypato-toxicity and poor solubility in water limits its clinical use. Two GDM derivatives, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) and 17-dimethylamino-ethylamino-17-demethoxygeldanamycin (17-DMAG), both showing lesser hepato-toxicity, are now in Phase II and Phase I clinic trials. In order to accomplish the structure modification of GDM by genetic means, an attempt to obtain the biosynthetic gene cluster of GDM from S. hygroscopicus 17997 was made. In this study, a pair of primers was designed according to a conserved sequence of one of possible post-PKS (polyketides synthase) modification genes, the carbamoyltransferase (CT) gene (gdmN) in GDM biosynthesis. The 732 bp PCR product was obtained from the S. hygroscopicus 17997 genomic DNA. Through the colony-PCR Binary Search Method, using the CT gene primers, six positive cosmid clones, CT1-6, were identified from the S. hygroscopicus 17997 cosmid genomic library. The CT gene containing fragments were verified and localized by Southern blot. The CT-4 positive cosmid was then sub-cloned and sequenced. Approximately 28.356kb of foreign gene sequence from CT-4 cosmid and by further PCR extension reaction was obtained. Based on BLAST analysis, this sequence contains 13 possible ORFs and their deduced functions are believed to be involved in GDM production. The ORF1 encoding products show homology (87%) with incomplete sixth module and complete seventh module of PKS, gdmA3, in S. hygroscopicus NRRL 3602. The ORF2-13 gene products are similar to gdmF(9 5%), gdmM(8 8%), gdmN (92%), gdmH (92%), I (93%), J (90%), K (93%), G (96%), gdmO (91%), gdmP (93%) and two transcription regulation genes gdmRI (83%) and gdmRII (90%). The obtained possible GDM biosynthetic gene cluster in S. hygroscopicus 17997 will facilitate the further functional analysis of the genes and to modify the structure of GDM through combinatorial biosynthesis.

Anti-Bacterial Agents↗
PubMed2002

Identification of a set of genes involved in the biosynthesis of the aminonucleoside moiety of antibiotic A201A from Streptomyces capreolus.

A novel cosmid (pABC6.5) whose DNA insert from Streptomyces capreolus, the A201A antibiotic producer, overlaps the inserts of the previously reported pCAR11 and pCAR13 cosmids, has been isolated. These two latter cosmids were known to contain the aminonucleoside antibiotic A201A resistance determinants ard2 and ard1, respectively. Together, these three cosmids have permitted the identification of a DNA stretch of 19 kb between ard1 and ard2, which should comprise a large region of a putative A201A biosynthetic (ata) gene cluster. The sequence of the 7 kb upstream of ard1 towards ard2 reveals seven consecutive open reading frames: ataP3, ataP5, ataP4, ataP10, ataP7, ata12 and ataPKS1. Except for the last two, their deduced products present high similarities to an identical number of counterparts from the pur cluster of Streptomyces alboniger that were either known or proposed to be implicated in the biosynthesis of the N6,N6-dimethyl-3'-amino-3'-deoxyadenosine moiety of puromycin. Because A201A contains this chemical moiety, these ataP genes are most likely implicated in its biosynthesis. Accordingly, the ataP4, ataP5 and ataP10 genes complemented specific puromycin nonproducing Deltapur4, Deltapur5 and Deltapur10 mutants of S. alboniger, respectively. Amino acid sequence comparisons suggest that ata12 and ataPKS1 could be implicated in the biosynthesis of the d-rhamnose and alpha-p-coumaric acid moieties of A201A. Further sequencing of 2 kb of DNA downstream of ard1 has disclosed a region which might contain one end of the ata cluster.

Amino Acid Sequence↗
PubMed1984

Cloned avirulence gene of Pseudomonas syringae pv. glycinea determines race-specific incompatibility on Glycine max (L.) Merr.

A genomic library of Pseudomonas syringae pv. glycinea race 6 DNA was constructed in the mobilizable cosmid vector pLAFR1 and maintained in Escherichia coli HB101. Completeness of the library was estimated by assaying clones for the expression of ice-nucleating activity in E. coli. Ice-nucleation activity was represented approximately once in every 600 clones. Six hundred eighty random race 6 cosmid clones were mobilized from E. coli by plasmid pRK2013 in individual conjugations to a race 5 strain of P. s. glycinea. A single clone (pPg6L3) was detected that changed the race specificity of race 5 from virulent (compatible) to avirulent (incompatible) on the appropriate soybean cultivars. The clone was also mobilized from E. coli into race 1 and race 4 strains of P. s. glycinea, and it conferred on these transconjugants the same host range incompatibility as the wild-type race 6 strain. The cosmid clone was mapped by restriction endonucleases, and two adjacent EcoRI fragments were identified by transposon Tn5 mutagenesis to be important in determining race specificity. Southern blot analysis showed that the two EcoRI fragments are unique to race 6 and are not present in the other races tested. The cosmid clone pPg6L3 was also mobilized to Pseudomonas fluorescens and Rhizobium japonicum. However, neither these isolates nor E. coli harboring pPg6L3 elicited a hypersensitive reaction in soybean leaves.

Journal Article↗
PubMed1988

Immunogenic (tum-) variants of mouse tumor P815: cloning of the gene of tum- antigen P91A and identification of the tum- mutation.

Mutagen treatment of mouse P815 tumor cells produces tum- variants that are rejected by syngeneic mice because these variants express new surface antigens. These "tum- antigens" are recognized by cytolytic T lymphocytes but induce no detectable antibody response. Transfection of P815 cell line P1.HTR with DNA of tum- variant P91 yielded transfectants expressing tum- antigen P91A. They were detected by their ability to stimulate proliferation of cytolytic T lymphocytes [Wölfel, T., Van Pel, A., De Plaen, E., Lurquin, C., Maryanski, J. L. & Boon, T. (1987) Immunogenetics 26, 178-187]. A cosmid library of a cell line expressing antigen P91A was transfected into P1.HTR. Transfectants expressing the antigen were obtained. By packaging directly the DNA of a transfectant with lambda phage extracts, we obtained a small cosmid population containing as major component a cosmid that transferred the expression of P91A. The assay of various restriction fragments of this cosmid led to the isolation of an 800-base-pair fragment containing the P91A sequence required for transfection. Comparison with a homologous cDNA showed that this fragment contained only one of the several exons of the P91A gene. The normal and the tum- forms of the gene differ by one nucleotide located in this 137-base-pair exon. The essential role of this mutation, which produces an amino acid change, was confirmed by site-directed mutagenesis. No significant sequence similarity was found between the 800-base-pair fragment and any recorded gene.

Amino Acid Sequence↗
PubMed1985

Characterization of DNA fragments encoding fimbriae of the uropathogenic Escherichia coli strain KS71.

Recombinant plasmids were constructed that expressed the KS71A, KS71B and KS71C fimbrial antigens of the pyelonephritogenic Escherichia coli strain KS71 (O4:K12) in E. coli HB101. The KS71C-encoding genes were located on a 6.4 kb HindIII-XhoI fragment obtained from the recombinant cosmid pKTH145 that expresses this antigen. Spontaneous KS71C-mutants were isolated that contained a 0.8 kb insert in a specific restriction fragment of KS71C-encoding recombinant plasmids. The KS71B-encoding segment was located on a 11.5 kb deletable DNA fragment of recombinant cosmid pKTH144. A DNA fragment encoding the KS71A fimbria was obtained on a 12 kb EcoRI fragment of the recombinant cosmid expressing this antigen in E. coli HB101 and closely resembled the KS71B-encoding fragment. In the recombinant cosmid, the KS71B-expressing region was flanked by homologous DNA segments. A similar stretch of DNA was found close to the KS71A-expressing DNA region.

Cloning, Molecular↗
PubMed1995

Complementation of Xanthobacter Py2 mutants defective in epoxyalkane degradation, and expression and nucleotide sequence of the complementing DNA fragment.

Three Xanthobacter Py2 mutants (M3, M8 and M10) lacking epoxyalkane-degrading activity were isolated and characterized. All mutants were able to grow on acetone, the degradation product of 1,2-epoxypropane conversions. Furthermore, they contained the unidentified 'low molecular mass fraction' (LMF) necessary for epoxyalkane-degrading activity. Three cosmids from a gene bank complemented the mutation in M10 and M8 but not in mutant M3. Epoxyalkane-degrading activity in crude extracts of 1,2-epoxypropane-grown complemented mutants was similar to the wild-type activity. Surprisingly, M10 transformed with complementing cosmid pEP9 showed a constitutively expressed epoxyalkane-degrading activity, which was not observed in the wild-type strain. The cosmid pEP9 was conjugated into Xanthobacter autotrophicus GJ10, which is not able to degrade 1,2-epoxypropane. In crude extracts of X. autotrophicus GJ10(pEP9), epoxyalkane-degrading activity was demonstrated, but only after the addition of the LMF from Xanthobacter Py2. Hybridization experiments demonstrated an overlap on complementing cosmids pEP1, pEP3 and pEP9. Subcloning revealed a 4.8 kb EcoRI-HindIII fragment to be necessary for complementing the mutant M10. In the sequence of this fragment four different ORFs were found.

Alkanes↗
PubMed1996

The genomic structure of discoidin receptor tyrosine kinase.

The discoidin domain receptor (DDR) is a new class of receptor tyrosine kinase that is distinguished by a unique extracellular domain homologous to the lectin Discoidin I found Dictyostelium discoideum. A cosmid was isolated from a human chromosome 6 cosmid library containing the DDR gene. A complete genomic contig of the DDR gene was constructed from seven subclones of the cosmid. The cosmid fragments were analyzed by PCR, sequencing, and comparison of genomic/cDNA sequence. The DDR gene is composed of 17 exons, ranging in size from 96 to 1014 bp, distributed along approximately 12 kb of genomic DNA. The extracellular domain is encoded by 8 exons of which three code for the discoidin domain. The transmembrane domain is encoded by 1 exon, the juxtamembrane by 3 exons, and the catalytic domain by 5 exons. The generation of the two splice variants of DDR, EDDR1 and EDDR2 are explained by the genomic structure. Exon 11 (111 bp in the juxtamembrane domain) is present in DDR and absent in the splice variant EDDR1. An inverted repeat of 20 bp was identified at the 3' exon-intron junction of exon 11, which results in a lariat loop-like secondary structure. EDDR2 is generated because of a cryptic splice acceptor site that results in an extra 18 bp (6 amino acids) inserted 5' of exon 14 in the catalytic domain. A polymorphic (GT)17 repeat was identified in intron 5 with a heterozygosity of 0.71. The exon-intron structure of the DDR gene will be helpful in further understanding of its function and explains the possible structural basis for the two splice variants.

Alternative Splicing↗
PubMed2005

The conserved Xanthomonas campestris pv. vesicatoria effector protein XopX is a virulence factor and suppresses host defense in Nicotiana benthamiana.

Nicotiana benthamiana leaves display a visible plant cell death response when infiltrated with a high titer inoculum of the non-host pathogen, Xanthomonas campestris pv. vesicatoria (Xcv). This visual phenotype was used to identify overlapping cosmid clones from a genomic cosmid library constructed from the Xcv strain, GM98-38. Individual cosmid clones from the Xcv library were conjugated into X. campestris pv. campestris (Xcc) and exconjugants were scored for an altered visual high titer inoculation response in N. benthamiana. The molecular characterization of the cosmid clones revealed that they contained a novel gene, xopX, that encodes a 74-kDa type III secretion system (TTSS) effector protein. Agrobacterium-mediated transient expression of XopX in N. benthamiana did not elicit the plant cell death response although detectable XopX protein was produced. Interestingly, the plant cell death response occurred when the xopX Agrobacterium-mediated transient expression construct was co-inoculated with strains of either XcvDeltaxopX or Xcc, both lacking xopX. The co-inoculation complementation of the plant cell death response also depends on whether the Xanthomonas strains contain an active TTSS. Transgenic 35S-xopX-expressing N. benthamiana plants also have the visible plant cell death response when inoculated with the non-xopX-expressing strains XcvDeltaxopX and Xcc. Unexpectedly, transgenic 35S-xopX N. benthamiana plants displayed enhanced susceptibility to bacterial growth of Xcc as well as other non-xopX-expressing Xanthomonas and Pseudomonas strains. This result is also consistent with the increase in bacterial growth on wild type N. benthamiana plants observed for Xcc when XopX is expressed in trans. Furthermore, XopX contributes to the virulence of Xcv on host pepper (Capsicum annuum) and tomato (Lycopersicum esculentum) plants. We propose that the XopX bacterial effector protein targets basic innate immunity in plants, resulting in enhanced plant disease susceptibility.

Amino Acid Sequence↗
PubMed1992

Molecular cloning of epithelial cell invasion determinants from enterotoxigenic Escherichia coli.

Although penetration of the epithelial mucosa has not been identified as a virulence mechanism in enterotoxigenic Escherichia coli (ETEC), we have found that this pathogen is capable of invading human intestinal cell lines. Classical ETEC strain H10407 was most invasive for epithelial cell lines derived from ileocecal and colonic tissues. An ETEC cosmid library was screened for clones that could direct E. coli HB101 to invade cultured human ileocecal epithelial cells (HCT 8 cells). Three invasive recombinant cosmid clones were isolated. These cosmids could direct HB101 invasion at an efficiency that was equal to or greater than that of the parent ETEC strain. The invasion cosmids also allowed for enhanced HCT 8 cell adherence by HB101. Electron micrographs of ETEC and recombinant HB101 strains revealed intracellular bacteria contained within endocytic vacuoles. Restriction endonuclease mapping and hybridization analyses showed that the three ETEC clones represent two separate invasion systems present in the parent ETEC strain and that both systems are chromosomally encoded. The parent ETEC strain and one cloned invasion system did not invade HeLa cells. Interestingly, one cloned invasion system was capable of directing HB101 to invade HeLa cells. Invasion of HCT 8 cells by recombinant HB101 strains and the parent ETEC strain was inhibited by cytochalasin D, indicating that the wild-type and both cloned invasion systems trigger an actin polymerization-dependent uptake process. It is not known whether the invasive phenotype of ETEC is relevant for enterotoxigenic disease. However, the parent ETEC strain, as well as recombinant HB101 strains, was capable of transcytosis through polarized HCT 8 monolayers. This transcytosis suggests that ETEC may cross the gut epithelium in vivo and that this invasion may have a previously unrecognized role in the disease process.

Animals↗
PubMed1994

Isolation and characterization of a gene involved in hemagglutination by an avian pathogenic Escherichia coli strain.

In this article, we report the isolation and characterization of a gene that may be important in the adherence of avian pathogenic Escherichia coli to the avian respiratory tract. The E. coli strain HB101, which is unable to agglutinate chicken erythrocytes, was transduced with cosmid libraries from the avian pathogenic E. coli strain chi 7122. Enrichment of transductants that could agglutinate chicken erythrocytes yielded 19 colonies. These isolates contained cosmids that encompassed four nonoverlapping regions of the E. coli chromosome. Only one group of cosmids, represented by pYA3104, would cause E. coli CC118 to agglutinate chicken erythrocytes. A 10-kb fragment of this cosmid was subcloned in pACYC184. Transposon mutagenesis of this fragment with Tn5seq1 indicated that a contiguous 4.4-kb region of cloned DNA was required for hemagglutination. In vitro transcription/translation assays indicated that this 4.4-kb region of DNA encoded one protein of approximately 140 kDa. The nucleotide sequence of this region was determined and found to encode one open reading frame of 4,134 nucleotides that would encode a protein of 1,377 amino acids with a deduced molecular weight of 148,226. This gene confers on E. coli K-12 a temperature-sensitive hemagglutination phenotype that is best expressed when cells are grown at 26 degrees C, and we have designated this gene tsh and the deduced gene product Tsh. Insertional mutagenesis of the chromosomal tsh gene in chi 7122 had no effect on hemagglutination titers. The deduced protein was found to contain significant homology to the Haemophilus influenzae and Neisseria gonorrhoeae immunoglobulin A1 proteases. These data indicate that (i) a single gene isolated from the avian pathogenic E. coli strain chi 7122 will confer on E. coli K-12 a hemagglutination-positive phenotype, (ii) chi 7122 contains at least two distinct mechanisms to allow hemagglutination to occur, and (iii) the hemagglutinin Tsh has homology with a class of proteins previously not known to exist in E. coli.

Amino Acid Sequence↗