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

Abstract

A hemagglutinin with an Mr of 67,000 (pMGA) from Mycoplasma gaUlisepticum 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 ofEcoRI-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 * Corresponding author.

A 41-kDa variable surface protein of Mycoplasma gallisepticum has a counterpart in Mycoplasma imitans and Mycoplasma iowae

“…Nevertheless, pMG41 and pMI41 are the first reported examples of variable surface proteins in mycoplasmas which are not described as integral membrane proteins. All other described phase-variable mycoplasma antigens so far are integral membrane proteins that are membrane-anchored either via lipid [12, 14,17] or via hydrophobic transmembrane domains (191; D. Yogev, D. Menaker and R. Rosengarten, submitted).…”

Sequence polymorphisms within the pMGA genes and pMGA antigenic variants in Mycoplasma gallisepticum

“…1). Type A encodes a longer PRR similar to the pMGA1.2 gene in the S6 strain [5,6]. Type B encodes a shorter PRR similar to the published sequence of the pMGA gene expressed in the type strain PG31 [11].…”

“…Immunoblotting with the PhastSystem (Pharmacia-LKB, Uppsala, Sweden) was used to determine regions of the pMGA molecule recognized by mAbs [12]. The whole length puri¢ed pMGA1.1 protein, its fragments, cleaved either with protease V-8 (Glu-C) or trypsin, or a truncated recombinant pMGA (residues from 37 to about 300) were used for the analysis [4,5]. Mapping of the epitopes for mAbs was conducted with the SPOTS system (Genosys, Cambridge, UK).…”

“…We have observed that M. gallisepticum strain S6 B and its derivative IHB1 often synthesized pMGA lacking the epitope for mAb 71. Using the SPOTS system we have found that mAb 71 reacts with the peptide TNSDEPRSIS, corresponding to the residues 266^275 of the pMGA1.2 [5,6]. However, mAb 71 reacted Proteins of clones C6/20 (lanes a) and C6/29 (lanes b) were separated by isoelectric focusing in parallel lanes and assayed as follows: A: By mAb 86 (86), mAb 71 (71), pAb t pMGA (RtP) or mAb 12D10 (12D).…”

“…Locations of epitopes for pAb D1, pAb t pMGA and mAbs on the pMGA molecule. Top line: pMGA1.1 protein [4,5] and locations of the epitopes determined in this study. Dark boxes indicate the position of the epitope for the corresponding antibody.…”