Mycoplasma pneumoniae cytadhesin P1 was purified by monoclonal antibody affinity chromatography followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal 18-aminoacid sequence of P1 was determined and used to design two synthetic oligonucleotides, a 14-mer corresponding to amino acids 1 to 5 and an 18-mer corresponding to amino acids 7 to 12. These oligonucleotides served as hybridization probes for the identification of the P1 gene by Southern blot analysis ofM. pneumoniae DNA. The P1 gene was cloned into plasmid pUC19 and mapped by using appropriate restriction endonucleases. The DNA sequence of the entire P1 gene was determined by subcloning appropriate DNA fragments into bacteriophage M13 and sequencing the DNA by the dideoxy-chain-termination method. The P1 gene contains an open reading frame of 4,881 nucleotides coding for a protein of 1,627 amino acids with a calculated molecular weight of 176,288. Properties of the amino-terminal sequence suggest that protein P1 may be synthesized as a precursor with subsequent processing to a mature protein of a calculated molecular weight of 169,758. Potential antigenic sites were determined by hydrophilicity plots. A computer search revealed that part of the predicted P1 sequence is homologous to cytoskeletal keratin of mammalian species and human fibrinogen alpha chain precursor. These results demonstrate the uniqueness of P1 as a cytadhesin and virulence determinant.
Mycoplasma pneumoniae is the causative agent of primary atypical pneumonia in humans . The intimate association between M. pneumoniae and the host cell surface is prerequisite for the development and persistence of disease (1, 2). Protein P 1 has been designated the major adhesin mediating the attachment of M. pneumoniae to respiratory epithelium (3-6). M. pneumoniae organisms that lack P1 or are unable to densely cluster and properly position the adhesin at the mycoplasma tip are avirulent (3,7,8). We recently cloned and sequenced the cytadhesin P 1 gene and deduced the protein sequence to determine the primary structure of P 1 and assist in identification of its functional domains (9).In this report a XgtI l recombinant DNA expression library of M. pneumoniae is constructed and used to characterize the P1 domain involved in cytadherence . Clones expressing P 1 epitopes were identified by screening the library with two categories of anti-P 1 mAbs known to block M. pneumoniae attachment to erythrocytes and respiratory epithelium . Clones were sequenced and their position located in the P1 structural gene. Since adhesin P1 is a major immunogen of M.pneumoniae (10), these clones were also tested for their reactivity with acute and convalescent sera from M. pneumoniae-infected patients . This experimental approach permits the identification of antigenic and biofunctional determinants of adhesin P 1 . Materials and Methods Brief Definitive ReportXgtI l DNA arms and phage extracts were purchased from Promega Biotech (Madison, WI). Enzymes used for constructing the genomic library were from New England Biolabs (Beverly, MA); restriction enzymes were from Bethesda Research Laboratories (Gaithersburg, MD).M. pneumoniae strain M129-B16 genomic DNA was sheared and ligated to expression vector Xgt11 according to the procedures described by Young and Davis (11,12). The genomic library was screened with a pool of two anti-P1 mAbs designated 5B8 and 6E7, which recognize distinct epitopes and block M. pneumoniae cytadherence (13). The recombinant phages were grown and DNA was extracted as described by Maniatis et al. (14). The inserts from the different clones were subcloned into M13mp19 phage and sequenced by the dideoxy chain termination method of Sanger et al. (15).M. pneumoniae genomic DNA was cut with different restriction enzymes and transferred to nitrocellulose (NC) filters according to Southern (16). Hybridizations were performed
We have isolated cDNA clones encoding bovine pancreatic preproglucagon. Twenty-five putative preproglucagon clones were selected by screening 3,100 clones of a fetal bovine pancreas cDNA library with a synthetic oligodeoxynucleotide probe. The probe was a mixture of synthetic 17-base DNA oligomers constructed to correspond to the six carboxyl-terminal amino acids (residues 24-29) of mature glucagon. Restriction mapping of six of these clones suggested that they represented a single mRNA species. Primary sequence analysis of one clone containing a 1,200-base-pair DNA insert revealed that it contained an essentially fulllength copy of glucagon mRNA. Analysis of the cDNA suggested a protein coding sequence of 540 nucleotides and 5'-and 3'-untranslated regions of 90 and 471 nucleotides, respectively. This cDNA sequence encoded a 20-amino acid signal sequence followed by one for glicentin, a 69-amino acid polypeptide containing an internal glucagon moiety that has been found in porcine intestines. Glicentin is followed by two additional glucagon-like peptides, each flanked by paired basic amino acids (Lys, Arg) characteristic of prohormone processing. These polypeptide sequences show striking homology with those for glucagon and other members of the glucagon family of peptides.
The Mycoplasma pneumoniae cytadhesin P1 structural gene with flanking regions was labeled by nick translation and used as a probe to analyze gene copy nuimber in M. pneumoniae. Multiple bands of genomic DNA were hybridized by the probe. To establish what part of the P1 gene existed as multiple copies, the P1 gene and regions adjacent to the 3' and 5' ends were divided with restriction enzymes into 14 segments ranging in size from 174 to 651 base pairs. These pieces were purified on agarose gels, subcloned into pUC19, purified, labeled by nick translation, and used to probe the entire M. pneumoniae genome. Several regions near the middle and carboxy end of the P1 structural gene hybridized to single copies. The remaining P1 subclones hybridized to multiple bands under stringent hybridization conditions, indicating extensive homology with other parts of the M. pneumoniae genome. The singleversus multiple-copy nature of P1 structural gene domains is discussed.
The Mycoplasma pneumoniae cytadhesin P1 genes from two groups of clinical isolates that display restriction fragment length polymorphisms were cloned and sequenced. Within each group the nucleotide sequences were identical, but two major differences were detected between the groups. These two stretches of sequence divergence were located in multiple-copy regions of the Pl gene and resulted in considerable amino acid changes. Mycoplasma pneumoniae is a cell wall-less procaryote that colonizes the ciliated respiratory epithelium of humans and causes primary atypical pneumonia (5, 11). Mycoplasma attachment to the respiratory cell surface is a critical step in the infection process, and previous studies have established that a 170-kilodalton surface protein, P1, located at the tiplike structure of virulent M. pneumoniae mediates cytadherence (2, 9, 12). Mutants that lack P1 or cannot cluster P1 at the tip are noncytadhering and avirulent (2, 4, 18). During M. pneumoniae infection both humans and experimental animals mount a strong immunological response against P1 that correlates with resolution of the disease (19, 33). To elucidate structural and functional properties of the P1 cytadhesin, we cloned and sequenced the gene (30). The P1 structural gene was contained within a 5.6-kilobase (kb) EcoRI piece of DNA, and further analysis revealed that two-thirds of the P1 gene was multiple copies (29). Since repeated gene sequences of major surface antigens can be associated with antigenic variation in pathogenic microorganisms (24, 27, 28, 31), P1 subclones generated in our previous study (29) were used to probe a collection of M. pneumoniae strains and clinical isolates. Two distinct hybridization patterns were detected, suggesting variability in their P1 genes (6). To further understand the nature of these observations, the P1 genes from several representative isolates of M. pneumoniae were cloned and sequenced. M. pneumoniae M129-B16 (ATCC 29342 in its sixteenth passage) was isolated in 1968 from a patient with M. pneumoniae disease (20) and served as the standard wild-type strain. M. pneumoniae FH (ATCC 15531) was purchased from the American Type Culture Collection, Rockville, Md. (5). M. pneumoniae TW 7-5 was obtained from J. G. Tully (National Institute of Allergy and Infectious Diseases) and originated from a group of mycoplasma isolates obtained from military recruits in 1974 and 1975 (3, 34). M. pneumo
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