pMGA phenotypic variation in Mycoplasma gallisepticum occurs in vivo and is mediated by trinucleotide repeat length variation

Chickens were infected with a pathogenic strain of Mycoplasma gallisepticum, and the expression of pMGA, the major surface protein, was inferred by examination of colonies from ex vivo cells. Within 2 days postinfection, 40% of cells had ceased the expression of the original pMGA surface protein (pMGA1.1), and by day 6, the majority of recovered cells were in this category. The switch in pMGA phenotype which had occurred in vivo was reversible, since most colonies produced from ex vivo progenitors exhibited frequent pMGA1. 1(+) sectors. After prolonged in vivo habitation, increasing proportions of recovered cells gave rise to variant pMGA colonies which had switched from the expression of pMGA1.1 to another gene, pMGA1.2, concomitant with the acquisition of a (GAA)(12) motif 5' to its promoter. Collectively, the results suggest that changes in M. gallisepticum pMGA gene expression in vivo are normal, common, and possibly obligate events for successful colonization of the host. Surprisingly, the initial cessation of pMGA1.1 expression occurred in the absence of detectable pMGA antibodies and seemed to precede the adaptive immune response.     

Haemagglutinins of pathogenic avian mycoplasmas

The pathogenic avian mycoplasmas, Mycoplasma gallisepticum , Mycoplasma synoviae , Mycoplasma meleagridis , Mycoplasma iowae and Mycoplasma imitans , synthesize haemagglutinins that are immunogenic, variably expressed, surface proteins. The haemagglutinins of M. gallisepticum (pMGA), M. synoviae (VlhA) and M. imitans are lipoproteins, encoded by related multigene families that appear to have arisen by horizontal gene transfer. M. gallisepticum also has genes encoding cytadhesins in its genome but these are present as a single copies, while the pMGA gene family contains 30 to 70 genes. The switch in expression of distinct pMGA genes (e.g. pMGA1.1 to pMGA1.9) generates antigenic variation, which is thought to be important in immune evasion but also has significance in the preparation of M. gallisepticum antigens for serological diagnosis. In the majority of M. synoviae strains, post-translational cleavage of the VlhA protein generates an amino-terminal part (the lipoprotein MSPB) and a carboxyl-terminal part (MSPA), which mediates binding to erythrocytes. The 5' vlhA gene region, which encodes proline-rich repeats in the amino-terminal part of MSPB, is highly polymorphic among M. synoviae strains. Insertions or deletions in the part of vlhA encoding the proline-rich repeats cause MSPB length variation in different M. synoviae strains. Recombination between the 5' vlhA gene and pseudogenes in the genome generates changes in antigenic determinants in the carboxyl two-thirds of the MSPB molecule, and in MSPA, resulting in changes in the domains involved in the binding of M. synoviae to erythrocytes. Variant haemagglutinins of M. gallisepticum (pMGA1.7) and M. synoviae (diverse VlhA forms) share sequences that may be responsible for antigenic cross-reactions between M. gallisepticum and M. synoviae . Shared epitopes have been demonstrated using specific antibodies against MSPB that also recognize proteins of M. gallisepticum and of M. iowae (serotype N). Size and antigenic variants have also been reported for M. meleagridis and M. iowae proteins, but it is not known if these are their haemagglutinins. Advances in the molecular characterization of M. gallisepticum (pMGA, pvpA ) and M. synoviae ( vlhA ) genes and their sequencing in numerous strains is likely to enable significantly improved epidemiological studies and improved tracing of M. gallisepticum and M. synoviae strains in different flocks.     

Identification of a virulence-associated determinant, dihydrolipoamide dehydrogenase (lpd), in Mycoplasma gallisepticum through in vivo screening of transposon mutants

To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.     

Molecular cloning of a member of the gene family that encodes pMGA, a hemagglutinin of Mycoplasma gallisepticum.

A hemagglutinin with an M(r) of 67,000 (pMGA) from Mycoplasma gallisepticum S6 was purified by using monoclonal antibody affinity chromatography. Purified pMGA was treated with a number of enzymes, the resultant peptides were purified, and their amino acid sequence was determined by using an Applied Biosystems (model 471A) protein sequencer. The DNA sequence encoding two peptides was used to dictate the sequences of synthetic oligonucleotides which were used to screen a library of EcoRI-cut M. gallisepticum DNA in pUC18. A clone reactive to both probes was isolated and found to contain a recombinant insert of 10 kb. The clone was mapped by using restriction endonucleases and fragments subcloned into pUC18 for DNA sequencing. Analysis of part of the DNA sequence revealed an open reading frame containing 1,941 nucleotides which encoded 647 amino acids. The amino terminus was preceded by a putative leader sequence of 25 amino acids. A promoter region preceding the putative start codon GUG was also located. This gene would encode a mature protein of 67,660 Da. There were a number of differences between the predicted amino acid sequence and that determined by direct peptide sequencing. Also, two tryptic peptides of pMGA were not found in the DNA sequence. This suggested that the cloned gene did not encode pMGA but did encode a homolog (pMGA1.2). Furthermore, downstream of pMGA1.2 was a region of DNA encoding a leader sequence followed by an amino acid sequence with high homology to that encoded by the pMGA1.2 gene. The presence within M. gallisepticum of a family of pMGA genes is inferred from the DNA sequence and Southern transfer data. A possible role for this gene family in immune evasion is discussed.     

Characterization of a major hemagglutinin protein from Mycoplasma gallisepticum.

Mycoplasma gallisepticum cell membranes were used to immunize mice to produce monoclonal antibodies to cell surface proteins. Three monoclonal antibodies were chosen for further characterization. All three reacted in immunoblots with an M. gallisepticum protein band of M(r) approximately 67,000 (designated pMGA). By using immunoelectron microscopy, pMGA was shown to be located on the cell surface. When M. gallisepticum whole cells were treated with up to 250 micrograms of trypsin per ml for 30 min, the only major protein lost from the cell surface as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western immunoblot transfer was pMGA. Two of the pMGA-specific monoclonal antibodies inhibited hemagglutination of chicken erythrocytes by M. gallisepticum S6, suggesting a role for pMGA in the attachment of M. gallisepticum to chicken erythrocytes. Sequencing the amino terminus of pMGA yielded 17 amino acids with no significant homology with the Mycoplasma pneumoniae attachment protein P1 or any other protein in the GenBank, Swiss-Prot, and EMBL data bases.     

The GAA repeat expansion in intron 1 of the frataxin gene is related to the severity of cardiac manifestation in patients with Friedreich's ataxia

Friedreich's ataxia is an autosomal recessive disorder characterized by spinocerebellar degeneration. It is caused by an unstable GAA trinucleotide repeat expansion (>120 repeats) in the first intron of the frataxin gene on chromosome 9 (9q13) in both alleles. Concentric left ventricular hypertrophy has been recognized as the major cardiac manifestation of Friedreich's ataxia. Our aim was to investigate the influence of the frataxin repeat length on cardiac hypertrophy in patients with Friedreich's ataxia and in patients with hypertrophic and dilated cardiomyopathy. Thirty-one patients with Friedreich's ataxia, 86 patients with hypertrophic cardiomyopathy, 134 patients with dilated cardiomyopathy, and 32 healthy individuals without cardiac disease were analysed by electrocardiography and 2D-M-mode echocardiography. Then, the size of the frataxin repeat was determined by polymerase chain reaction (PCR) and agarose gel electrophoresis. The number of GAA repeats in patients with hypertrophic and dilated cardiomyopathy was not different from the length in patients without cardiac disease (hypertrophic cardiomyopathy, 8+/-2 repeats on GAA 1 allele and 11+/-5 repeats on GAA 2 allele; dilated cardiomyopathy, 7+/-2 repeats on GAA 1 allele and 11+/-5 repeats on GAA 2 allele; Control, 9+/-1 repeats on GAA 1 allele and 12+/-6 repeats on GAA 2 allele). The septal and posterior wall thickness of these patients was not related to the GAA repeat length. All patients with Friedreich's ataxia had two enlarged alleles with a mean GAA repeat length of 757+/-316 and 1012+/-231, respectively. The lengths of both alleles were significantly greater than the lengths in the controls (P<0.0001), patients with hypertrophic cardiomyopathy (P<0.0001) and dilated cardiomyopathy (P<0.0001). A significant correlation was revealed between interventricular septal hypertrophy and frataxin repeat length in the smaller allele. Furthermore, the ratio of septal to posterior wall thickness was significantly correlated to GAA repeat size on the smaller allele. In conclusion, the size of the GAA repeat on the smaller allele in the frataxin gene is associated with the degree of left ventricular hypertrophy in patients with Friedreich's ataxia but is not related to the severity of hypertrophic cardiomyopathy.     

Factors affecting long-term stability of Moloney murine leukemia virus-based vectors

We have examined the long-term functional and structural stability of retroviral vectors in infected murine cells. We have used Moloney murine leukemia virus-based vectors expressing human HPRT, firefly luciferase (luc), and Escherichia coli beta-galactosidase (lacZ) as reporter genes, and the human HPRT and the transposon Tn5 neomycin resistance (neo) gene as selectable markers. All vectors, whether single or double gene, yielded both stable and unstable clones. Stability of the proviruses was dependent on a number of factors, including the nature of the infected cell, the reporter gene, the integration site of the provirus, the relative positions of the component genes in multigene vectors, and the presence or absence of selection pressure. Selection pressure was helpful, but not universally effective, in maintaining provirus structural and functional integrity. Reporter gene expression from an internal promoter was likely to be unstable with or without selection for an upstream, LTR-driven neo gene. In some clones, loss of proviral gene expression was accompanied by deletions, while other inactive clones retained an apparently intact provirus. In the latter clones, treatment with 5-azacytidine failed to reactivate the reporter genes, but superinfection with helper virus resulted in the reappearance of transmissible vector, indicating a reversible epigenetic mechanism for proviral shutdown. The design of effective retroviral vectors and their possible use in vivo will require further characterization of these determinants of provirus stability.     

Tandem repeat sequence variation as causative cis-eQTLs for protein-coding gene expression variation: the case of CSTB

Association studies have revealed expression quantitative trait loci (eQTLs) for a large number of genes. However, the causative variants that regulate gene expression levels are generally unknown. We hypothesized that copy-number variation of sequence repeats contribute to the expression variation of some genes. Our laboratory has previously identified that the rare expansion of a repeat c.-174CGGGGCGGGGCG in the promoter region of the CSTB gene causes a silencing of the gene, resulting in progressive myoclonus epilepsy. Here, we genotyped the repeat length and quantified CSTB expression by quantitative real-time polymerase chain reaction in 173 lymphoblastoid cell lines (LCLs) and fibroblast samples from the GenCord collection. The majority of alleles contain either two or three copies of this repeat. Independent analysis revealed that the c.-174CGGGGCGGGGCG repeat length is strongly associated with CSTB expression (P = 3.14 × 10(-11)) in LCLs only. Examination of both genotyped and imputed single-nucleotide polymorphisms (SNPs) within 2 Mb of CSTB revealed that the dodecamer repeat represents the strongest cis-eQTL for CSTB in LCLs. We conclude that the common two or three copy variation is likely the causative cis-eQTL for CSTB expression variation. More broadly, we propose that polymorphic tandem repeats may represent the causative variation of a fraction of cis-eQTLs in the genome.     

Using reporter genes to label selected neuronal populations in transgenic mice for gene promoter, anatomical, and physiological studies

This review summarizes recent work on the use of reporter genes to label selected neuronal populations in transgenic mice, with particular emphasis on gonadotropin-releasing hormone (GnRH) neurons. Reporter genes discussed are the lacZ, green fluorescent protein (GFP), luc, and bla genes, which encode the reporter proteins beta-galactosidase, GFP, luciferase, and beta-lactamase, respectively. Targeted transgenic expression of these reporter proteins is obtained by fusing the corresponding reporter gene, with or without a subcellular localization signal, to a cell type- or brain region-specific gene promoter. Mice carrying GnRH promoter-driven reporter genes have proven useful for revealing the promoter elements required for cell type-specific expression of GnRH, the full anatomical profile of the GnRH neuronal network, and its electrophysiological activity, suggesting that similar approaches will assist in elucidating the properties of other neuronal populations as well.     

Molecular Analysis of GAA Repeats and Four Linked Bi-allelic Markers in and Around the Frataxin Gene in Patients and Normal Populations from India

Friedreich ataxia (FRDA), the most common type of ataxia worldwide, is an autosomal recessive disease. Homozygous expansion of GAA repeats in the first intron of the frataxin gene constitute the major type of mutation that causes the disease. The prevalence of FRDA in diverse ethnic populations of India has not been widely studied. We have studied the distribution of polymorphic GAA repeats in the frataxin gene among 6 clinically diagnosed patients and 160 ethnically matched normal individuals, to gather information on the prevalence of FRDA in the eastern part of India. Homozygous expansion in the range of 250–730 GAA repeats was detected among the patients. Among normal individuals, we observed a unimodal distribution of GAA repeats, consisting of 10 different alleles ranging from 7 to 16 GAA repeats, where the 9 repeat allele had maximal frequency. Only 5.9% of all chromosomes were found to harbour >12 GAA repeats. Haplotype analysis using closely linked four bi‐allelic markers in and around the frataxin gene indicated that 66.7% of the expanded alleles harbour the ATCC haplotype that has been reported worldwide. This haplotype was present in 53.3% of the chromosomes with >12 GAA repeats, and accounted for only 3.8% of chromosomes with 7 to 12 GAA repeats. We found one novel haplotype, ACCT, among the expanded alleles as well as among normal individuals, though at low frequency; this haplotype may be characteristic of Indian populations.     

Mouse dystrophin enhancer preferentially targets lacZ expression in skeletal and cardiac muscle.

Duchenne muscular dystrophy is a muscle wasting disease that results from a dystrophin deficiency in skeletal and cardiac muscle. Studies concerning the regulatory elements that govern dystrophin gene expression in skeletal and/or cardiac muscle in both mouse and human have identified a promoter and an enhancer located in intron 1. In transgenic mice, the muscle promoter alone targets the expression of a lacZ reporter gene only to the right ventricle of the heart, suggesting the need for other regulatory elements to target skeletal muscle and the rest of the heart. Here we report that the mouse dystrophin enhancer from intron 1 can target the expression of a lacZ reporter gene in skeletal muscle as well as in other heart compartments of transgenic mice. Our results also suggest that sequences surrounding the mouse dystrophin enhancer may affect its function throughout mouse development.     

Autonomous expression of the slo gene of the bicistronic nga-slo operon of Streptococcus pyogenes

A recent model for cytolysin-mediated translocation in Streptococcus pyogenes proposes that NAD-glycohydrolase is translocated through streptolysin O-generated pores into a host cell (J. Madden, N. Ruiz, and M. Caparon, Cell 104:143-152, 2001). This model also assumes that the NAD-glycohydrolase (nga) and streptolysin O (slo) genes that code for these products are organized in an operon-like structure expressed from a single promoter only (nga). We expand this model by showing that slo possesses its own autonomous promoter, which is located 155 bp upstream of the slo gene. Under experimental conditions in which S. pyogenes is grown in THY medium, the strength of the slo promoter, as measured by the activity of a lacZ reporter gene, resulted in low but highly reproducible values. Finally, we demonstrated that sloR, a S. pyogenes gene that closely resembles the Clostridium perfringens pfoR gene, exerts a negative effect on the expression of the slo gene.