Sequence analysis of the major outer membrane protein gene of an ovine abortion strain of Chlamydia psittaci

The major outer membrane protein (MOMP) gene from an ovine abortion strain of Chlamydia psittaci (S26/3) has been cloned and sequenced. The gene shows the features of other chlamydial MOMPs but comparison with the previously reported sequence for the ovine abortion isolate A22/M has revealed substantial sequence divergence which is clustered into the same four intramolecular regions as the sequence variation found between C. trachomatis serovars. Subsequent restriction enzyme analysis of A22/M DNA has shown that it has an avian-type genomic profile and thus the comparison is between types rather than between strains.     

Genetic diversity of avian and mammalian Chlamydia psittaci strains and relation to host origin.

Genetic relationships were reported for Chlamydia psittaci derived from psittacine birds, pigeons, turkeys, humans, cats, muskrats, cattle, and sheep and for C. trachomatis, including representative strains of the three biovars, through physical analysis of genomic DNA including DNA fingerprinting with restriction endonuclease SalI, DNA-DNA hybridization in solution with S1 nuclease, and Southern analysis with genomic DNA probes. A total of 26 strains were divided into four groups of C. psittaci and two groups of C. trachomatis, on the basis of DNA fingerprints. The six groups of Chlamydia spp. were related to host origin: two avian groups (Av1 and Av2), one feline and muskrat group (Fe1), one ruminant group (Ru1), one C. trachomatis biovars trachoma and lymphogranuloma group (CtHu), and one C. trachomatis mouse biovar group (CtMo), although an ovine abortion strain belonged to the avian group Av2. DNA-DNA hybridization assay and Southern analysis with genomic DNA probes indicated three DNA homology groups in the genus Chlamydia: an avian-feline group (groups Av1, Av2, and Fe1), a ruminant group (group Ru1), and a C. trachomatis group (groups CtHu and CtMo). Furthermore, the Southern analysis indicated that the homologous sequences (DNA homology of at least 14%) within the avian-feline group were distributed along the whole genome, whereas the homologous sequences (DNA homology of less than 24%) among the three DNA homology groups were localized in distinct regions of the genome DNA. These results suggest that Chlamydia spp. are derived from a common ancestor and have diverged into various groups showing restricted host ranges as a natural characteristic and that the species C. psittaci should be differentiated into groups related to host origin and DNA homology.     

Chlamydia psittaci ewe abortion agent: complete nucleotide sequence of the major outer membrane protein gene

The complete nucleotide sequence encoding the major outer membrane protein (MOMP) of Chlamydia psittaci strain A22/M, responsible for enzootic abortion of ewes (EAE), has been determined. An 800bp Eco RI/ Xba I fragment containing a portion of the MOMP coding sequence from C. trachomatis serovar L1 was used to probe a λL47.1 genomic library constructed from DNA obtained from C. psittaci EAE A22/M. The recombinant L47.1/EA1 was selected and contained the entire C. psittaci MOMP gene within a 7.5 kb Bam HI fragment. The DNA sequence revealed an open reading frame encoding 402 amino acids, including a 22 amino acid signal peptide, which exhibited 17/22 conservation with the signal peptide of C. trachomatis MOMP. The calculated molecular mass of the C. psittaci MOMP was 43 kDa. A comparison of the MOMP genes of C. psittaci and C. trachomatis revealed only 34% nucleotide sequence homology, but 65% amino acid homology.     

Plasmids of Chlamydia psittaci: Cloning and comparison of isolates by Southern hybridisation

Abstract A chlamydial plasmid, 6.2 kb in size, was isolated from an avian strain of Chlamydia psittaci and cloned into the Eco RI site of pUC13. A restriction enzyme cleavage map of the resultant clone, pAP¹p, was very similar to the published map of the plasmid cloned from the C. psittaci meningopneumonitis strain Cal-10. Southern hybridisation analyses using pAP¹p as a probe, revealed the presence of plasmids with homologous DNA sequences in avian psittacosis, avian ornithosis, ovine polyarthritis and sporadic bovine encephalomyelitis strains of C. psittaci , as well as the LGV strain of Chlamydia trachomatis . Plasmid was not detected in koala conjunctivitis, ovine abortion or feline conjunctivitis isolates. The plasmid-containing isolates could be grouped according to size (6.2 or 7.2–7.3 kb) and restriction endonuclease pattern. These three plasmid categories correlate with previously reported C. psittaci biotypes, immunotypes and serotypes. The absence of plasmid from three infectious, pathogenic strains of C. psittaci suggests that, in this species at least, plasmid-encoded genes are not essential for survival, infectivity or virulence of the parasite.     

Cloning and antigenic expression of two genes from Chlamydia psittaci avian strain

Abstract: Genes from Chlamydia psittaci P-1041 were cloned into the Bam HI site of pUC19 and were transformed to host Escherichia coli JM109. Two recombinant plasmids that expressed protein antigens of Chlamydia were isolated. The sizes of the DNA fragments were 1350 and 1710 bp, and encoded for polypeptides of M _r 25 and 42 kilodaltons (kDa), respectively. The 25-kDa protein had cross-reactivity with antisera to ten C. psittaci strains and two C. trachomatis strains, whereas the 42-kDa protein reacted only with homologous antiserum to the C. psittaci P-1041 strain. Furthermore, in Southern hybridization analysis these two fragments as probes hybridized with DNA of ten C. psittaci strains and four C. trachomatis strains. These results indicated that the two fragments shared a DNA sequence common to the chlamydial genus.     

Genome sequence of the cat pathogen, Chlamydophila felis.

Chlamydophila felis (Chlamydia psittaci feline pneumonitis agent) is a worldwide spread pathogen for pneumonia and conjunctivitis in cats. Herein, we determined the entire genomic DNA sequence of the Japanese C. felis strain Fe/C-56 to understand the mechanism of diseases caused by this pathogen. The C. felis genome is composed of a circular 1,166,239 bp chromosome encoding 1005 protein-coding genes and a 7552 bp circular plasmid. Comparison of C. felis gene contents with other Chlamydia species shows that 795 genes are common in the family Chlamydiaceae species and 47 genes are specific to C. felis. Phylogenetic analysis of the common genes reveals that most of the orthologue sets exhibit a similar divergent pattern but 14 C. felis genes accumulate more mutations, implicating that these genes may be involved in the evolutional adaptation to the C. felis-specific niche. Gene distribution and orthologue analyses reveal that two distinctive regions, i.e. the plasticity zone and frequently gene-translocated regions (FGRs), may play important but different roles for chlamydial genome evolution. The genomic DNA sequence of C. felis provides information for comprehension of diseases and elucidation of the chlamydial evolution.     

Structures of and allelic diversity and relationships among the major outer membrane protein (ompA) genes of the four chlamydial species.

DNA sequences coding for 81% of the ompA gene from 24 chlamydial strains, representing all chlamydial species, were determined from DNA amplified by polymerase chain reactions. Chlamydial strains of serovars and strains with similar chromosomal restriction fragment length polymorphism had identical ompA DNA sequences. The ompA sequences were segregated into 23 different ompA alleles and aligned with each other, and phylogenetic relationships among them were inferred by neighbor-joining and maximum parsimony analyses. The neighbor-joining method produced a single phylogram which was rooted at the branch between two major clusters. One cluster included all Chlamydia trachomatis ompA alleles (trachoma group). The second cluster was composed of three major groups of ompA alleles: psittacosis group (alleles MN, 6BC, A22/M, B577, LW508, FEPN, and GPIC), pneumonia group (Chlamydia pneumoniae AR388 with the allele KOALA), and polyarthritis group (ruminant and porcine chlamydial alleles LW613, 66P130, L71, and 1710S with propensity for polyarthritis). These groups were distinguished through specific DNA sequence signatures. Maximum parsimony analysis yielded two equally most parsimonious phylograms with topologies similar to the ompA tree of neighbor joining. Two phylograms constructed from chlamydial genomic DNA distances had topologies identical to that of the ompA phylogram with respect to branching of the chlamydial species. Human serovars of C. trachomatis with essentially identical genomes represented a single taxonomic unit, while they were divergent in the ompA tree. Consistent with the ompA phylogeny, the porcine isolate S45, previously considered to be Chlamydia psittaci, was identified as C. trachomatis through biochemical characteristics. These data demonstrate that chlamydial ompA allelic relationships, except for human serovars of C. trachomatis, are cognate with chromosomal phylogenies.     

Physical mapping of yeast artificial chromosomes containing sequences from the human β-globin gene region

The recently developed technique for cloning genomic DNA fragments of several hundred kilobases or more into yeast artificial chromosomes (YACs) makes it possible to isolate gene families while preserving their structural integrity. We have analyzed five independent yeast clones identified by PCR screening using oligonucleotides derived from the adult human β-globin gene. Analysis of the five clones containing YACs by conventional and pulsed-field gel electrophoresis revealed that all of the clones include a YAC with sequences from the adult β-globin gene as expected. One of the clones contains multiple, unstable YACs. Two other clones carry single YACs in which there are at least two unrelated human genomic inserts. The remaining two clones contain single YACs, 150 and 220 kb in size, that contain the entire β-globin gene family and flanking regions in a single, structurally intact genomic fragment. These should prove useful in future studies of the regulation of expression of genes in the β-globin gene cluster.     

Development of a simplified polymerase chain reactionenzyme immunoassay for the detection of Chlamydia pneumoniae

The 16S rRNA genes of two Chlamydia pneumoniae and two C. psittaci strains of different serovars were sequenced then compared to previously reported Chlamydia 16S rRNA gene sequences. Chlamydia pneumoniae -specific regions were identified and specific primers for nested PCR were synthesized. Nested PCR reactions were performed, in a single tube, by varying the annealing temperature of the amplification cycles. The initial thermal cycles were selected to allow annealing and extension of only the outer primer pair, whilst in later cycles a temperature that allowed inner primer annealing was employed. The inner primers were labelled, one with biotin and the other with fluorescein and consequently the dual labelled amplicon could be immobilized onto antibiotin-coated microtitre plates and detected colorimetrically via an antifluorescein-enzyme conjugate. The assay was found to be sensitive and specific. No cross reactions were observed with C. trachomatis, C. psittaci or other common respiratory pathogens.     

Schistosoma japonicum: analysis of eggshell protein genes, their expression, and comparison with similar genes from other schistosomes.

As the egg of Schistosoma japonicum plays a central role in transmission and in pathogenesis, we sought to understand the molecular biology of egg formation. In this study we characterized an eggshell protein gene of S. japonicum and compared it with similar genes from S. mansoni and S. haematobium. To initiate studies on the eggshell protein genes of S. japonicum, a cloned genomic fragment containing an entire copy of a S. haematobium eggshell protein gene was used to identify three EcoRI hybridizing fragments of 2.6, 2.0, and 1.3 kbp in S. japonicum genomic DNA and to isolate three independent genomic clones from a S. japonicum genomic library. Two genomic clones, SJ 4-1 and SJ 3-1, contain at least two copies of the gene. The DNA sequence of a 2.0-kbp EcoRI fragment of clone SJ 3-1 showed two open reading frames (ORF), one of which showed a strong homology to the chorion proteins of insects. This ORF had 207 amino acids with a calculated molecular size of 18.5 kDa. The predicted peptide was glycine (50%) and tyrosine (10%) rich like other described schistosome eggshell proteins. Primer extension and the dideoxynucleotide sequence of the mRNA defined the cap site of the RNA and positioned the putative TATA and CAAAT elements and other cis-acting elements. Northern analysis demonstrated that eggshell protein mRNA was only detected in mature female parasites. The appearance of the female-specific mRNA was dependent on pairing with the male parasite and increased with egg production (as determined by hybridization intensity). A comparison of the DNA and deduced protein sequences of eggshell protein genes from S. japonicum with those of similar genes from S. mansoni and S. haematobium indicated that the genes are highly conserved, with S. mansoni and S. haematobium genes being more similar to each other than either is to S. japonicum.     

Physical mapping of yeast artificial chromosomes containing sequences from the human beta-globin gene region

The recently developed technique for cloning genomic DNA fragments of several hundred kilobases or more into yeast artificial chromosomes (YACs) makes it possible to isolate gene families while preserving their structural integrity. We have analyzed five independent yeast clones identified by PCR screening using oligonucleotides derived from the adult human beta-globin gene. Analysis of the five clones containing YACs by conventional and pulsed-field gel electrophoresis revealed that all of the clones include a YAC with sequences from the adult beta-globin gene as expected. One of the clones contains multiple, unstable YACs. Two other clones carry single YACs in which there are at least two unrelated human genomic inserts. The remaining two clones contain single YACs, 150 and 220 kb in size, that contain the entire beta-globin gene family and flanking regions in a single, structurally intact genomic fragment. These should prove useful in future studies of the regulation of expression of genes in the beta-globin gene cluster.     

A pseudogene homologous to mouse transplantation antigens: Transplantation antigens are encoded by eight exons that correlate with protein domains

We have isolated about 30 to 40 different BALB/c mouse sperm DNA genomic clones that hybridize to cDNA clones encoding proteins homologous to transplantation antigens. One of these clones (27.1) was selected for sequence analysis because it was polymorphic in Southern blot analyses of the DNAs from BALB/c and CBA mice. A fragment of 5.7 kilobases of this clone was completely sequenced and found to contain a pseudogene whose sequence is highly homologous to the sequences of known transplantation antigens. Pseudogene 27.1 is split into eight exons that correlate with the structurally defined protein domains of transplantation antigens. Using Southern blot hybridization on the DNAs of different inbred mouse strains, we mapped the pseudogene to the Qa-2,3 region, a part of the Tla complex on chromosome 17 that is adjacent to the major histocompatibility complex. The Qa-2,3 region encodes lymphoid differentiation antigens homologous to the transplantation antigens in size, in peptide map profiles and in their association with β2-microglobulin. These mapping studies suggest that gene 27.1 may be a pseudogene for either a Qa antigen or an as yet undefined transplantation antigen. Accordingly, we may have isolated genes encoding lymphoid differentiation antigens of the Tla complex as well as those encoding transplantation antigens among the 30 to 40 different genomic clones isolated from our sperm library.     

Analysis by molecular cloning of the human class II genes

The HLA class II genes control immune responsiveness to defined antigens; they encode cell surface heterodimers composed of alpha and beta glycopeptides. Recently, cDNA and genomic clones encoding these chains have been isolated, which allows molecular analysis of the class II genes. cDNA clones encoding the alpha chain of the HLA-DR antigen as well as that of another HLA class II antigen have been identified and characterized by nucleotide sequence analysis. These clones have been used as probes to isolate additional class II alpha cDNA clones in cDNA libraries and to identify polymorphisms in genomic DNA. Polymorphic restriction sites have been localized within the HLA-DR alpha gene and used as genetic markers in the analysis of families and of disease (insulin-dependent diabetes mellitus) and control populations. In addition, cDNA clones encoding the DR beta and DC beta chains were used as hybridization probes to identify DNA polymorphism. cDNA clones encoding the DR gamma (Ii) chain have also been identified; unlike the DR alpha and DR beta loci, the DR gamma gene is located on some chromosome other than chromosome 6. The genetic complexity of the human class II alpha and beta loci, as revealed by analysis with cDNA and genomic clones, is greater than that of the murine class II genes. The extent of that complexity will be defined by future work in this area.     

Evolutionary relationships among members of the genus Chlamydia based on 16S ribosomal DNA analysis.

Nucleotide sequences from strains of the four species currently in the genus Chlamydia, C. pecorum, C. pneumoniae, C. psittaci, and C. trachomatis were investigated. In vitro-amplified RNA genes of the ribosomal small subunit from 30 strains of C. pneumoniae and C. pecorum were subjected to solid-phase DNA sequencing of both strands. The human isolates of C. pneumoniae differed in only one position in the 16S rRNA gene, indicating genetic homogeneity among these strains. Interestingly, horse isolate N16 of C. pneumoniae was found to be closely related to the human isolates of this species, with a 98.9% nucleotide similarity between their 16S rRNA sequences. The type strain and koala isolates of C. pecorum were also found to be very similar to each other, possessing two different 16S rRNA sequences with only one-nucleotide difference. Furthermore, the C. pecorum strains truncated the 16S rRNA molecule by one nucleotide compared to the molecules of the other chlamydial species. This truncation was found to result in loss of a unilaterally bulged nucleotide, an attribute present in all other eubacteria. The phylogenetic structure of the genus Chlamydia was determined by analysis of 16S rRNA sequences. All phylogenetic trees revealed a distinct line of descent of the family Chlamydiaceae built of two main clusters which we denote the C. pneumoniae cluster and the C. psittaci cluster. The clusters were verified by bootstrap analysis of the trees and signature nucleotide analysis. The former cluster contained the human isolates of C. pneumoniae and equine strain N16. The latter cluster consisted of C. psittaci, C. pecorum, and C. trachomatis. The members of the C. pneumoniae cluster showed tight clustering and strain N16 is likely to be a subspecies of C. pneumoniae since these strains also share some antigenic cross-reactivity and clustering of major outer membrane protein gene sequences. C. psittaci and strain N16 branched early out of the respective cluster, and interestingly, their inclusion bodies do not stain with iodine. Furthermore, they also share less reliable features like normal elementary body morphology and plasmid content. Therefore, the branching order presented here is very likely a true reflection of evolution, with strain N16 of the species C. pneumoniae and C. psittaci forming early branches of their respective cluster and with C. trachomatis being the more recently evolved species within the genus Chlamydia.     

Construction and characterization of the transformation-competent artificial chromosome (TAC) libraries of Leymus multicaulis

Transformation-competent artificial chromosome system is able to clone and transfer genes efficiently in plants.In order to clone genes highly tolerant to barley yellow dwarf virus(BYDV),Aphids,drought and salt from Leymus multicaulis,the two TAC genomic libraries I and II were constructed in vector pYLTAC17 and pYLTAC747H/sacB,which contain about 165000 and 236000 recombinant clones sepa-rately.The genome coverage of the two libraries was totally estimated to be about 3―5 haploid genome equivalents,as size selection of genomic DNA fragments was approximately from 9 to 300 kb.Clones of the genomic libraries were collected as bulked pools each containing 500 clones or so,stored in twelve 96-deep-well plates and then were gridding in triplicate onto a high-density colony hybridization filter with a 3×3 pattern using a GeneTAC?G3 arraying robot after being transferred manually into three 384-well plates.Meanwhile 2501 and 2890 clones of Library in pYLTAC17 and in pYLTAC747H/sacB were stored individually in fourteen 384-well plates and then were automatically gridding in duplicate onto a high-density colony hybridization filter with a 6×6 pattern after a replication of plates.Nineteen positive clones were detected by using the probe glutahione reductase gene of L.multicaulis.TAC libraries constructed here can be used to isolate genomic clones containing target genes,and to carry out genome walking for positional cloning.Once the target TAC clones were isolated,they could be immediately transferred into plant genomes with the Agrobacterium system.