Complete sequence analysis of the genome of the bacterium Mycoplasma pneumoniae

The entire genome of the bacterium Mycoplasma pneumoniae M129 has been sequenced. It has a size of 816,394 base pairs with an average G+C content of 40.0 mol%. We predict 677 open reading frames (ORFs) and 39 genes coding for various RNA species. Of the predicted ORFs, 75.9% showed significant similarity to genes/proteins of other organisms while only 9.9% did not reveal any significant similarity to gene sequences in databases. This permitted us tentatively to assign a functional classification to a large number of ORFs and to deduce the biochemical and physiological properties of this bacterium. The reduction of the genome size of M. pneumoniae during its reductive evolution from ancestral bacteria can be explained by the loss of complete anabolic (e.g. no amino acid synthesis) and metabolic pathways. Therefore, M. pneumoniae depends in nature on an obligate parasitic lifestyle which requires the provision of exogenous essential metabolites. All the major classes of cellular processes and metabolic pathways are briefly described. For a number of activities/functions present in M. pneumoniae according to experimental evidence, the corresponding genes could not be identified by similarity search. For instance we failed to identify genes/proteins involved in motility, chemotaxis and management of oxidative stress.     

Systematic functional analysis of the yeast genome

The genome sequence of the yeast Saccharomyces cerevisiae has provided the first complete inventory of the working parts of a eukaryotic cell. The challenge is now to discover what each of the gene products does and how they interact in a living yeast cell. Systematic and comprehensive approaches to the elucidation of yeast gene function are discussed and the prospects for the functional genomics of eukaryotic organisms evaluated.     

Functional analysis of glycosyltransferases encoded by the capsular polysaccharide biosynthesis locus of Streptococcus pneumoniae serotype 14

Bacteria belonging to the species Streptococcus pneumoniae vary in their capsule. Presently, 90 capsular serotypes are known, all possessing their own specific polysaccharide structure. Little is known about the biosynthesis of these capsular polysaccharides. The cps locus of S. pneumoniae serotype 14 was cloned. So far, 7 open reading frames have been sequenced, cps14B to cps14H. The gene products are similar to proteins involved in bacterial polysaccharide biosynthesis, both of Gram-negative and -positive micro-organisms. Gene-specific mutants were created for cps14D to cps14H by insertional mutagenesis. All mutants no longer agglutinated with a monoclonal antibody against type 14 capsule polysaccharides. The biosynthetic function of cps14E and cps14G was determined by analysis of the intermediates in the synthesis of the oligosaccharide subunit, formed in membrane preparations of the wild-type and mutant strains and in membrane preparations of Escherichia coli expressing the pneumococcal glycosyltransferases. The enzyme encoded by cps14E is a glucosyl-1-phosphate transferase that links glucose to a lipid carrier, the first step in the biosynthesis of the type 14 repeating unit. The gene product of cps14G encodes a beta-1,4-galactosyltransferase, the enzyme responsible for the second step in the subunit synthesis, the transfer of galactose to lipid-linked glucose.     

Changes in the epidemiological pattern of Mycoplasma pneumoniae pneumonia

The aim of this clinical study was to investigate the changes in the epidemiological pattern and clinical characteristics of Mycoplasma pneumoniae pneumonia (MP). 244 patients with MP admitted to Kamo Hospital from 1979 to 1996 were reviewed. In the first half (1979-1987) there was a sharp increases once every 4-5 years in 1979-1978, 1984-1985, and 1988, but in the latter half (1988-1996) there were not sharp increases and decreases. The latest MP incidence in winter (Dec. to Feb.) was 24% in the latter half (1988-1996) significantly (p < 0.05) higher than 13% in the first half (1988-1996). In recent years in the management of pneumonia we should take MP into consideration every year and every season.     

Mycoplasma pneumoniae detection from throat swab by two-step polymerase chain reaction

Two-step polymerase chain reaction (PCR) with primers designated against 16S rRNA gene of Mycoplasma pneumoniae for diagnosis of infection was evaluated in comparison with the conventional single-step PCR and culture methods. The two-step PCR method showed specific amplification of M. pneumoniae DNA and higher sensitivity (1.5 fg/assay) than the single-step PCR method. With the two-step PCR method, 76 of 322 throat swabs (23.6%) from patients with acute respiratory complaints gave positive results whereas 20.2% were positive in the culture method. Seven of 13 samples which were negative in the single-step PCR method but positive in either serological or the culture method showed positive results by the two-step PCR method. In addition, 5 samples which were weakly positive in the single-step PCR method showed distinctly positive results in the two-step PCR. These results indicate that the two-step PCR method is a useful tool for detection of M. pneumoniae in clinical specimens, although it requires a relatively sophisticated in technique.     

Inverted repetitive sequences in the human genome

A specific class of DNA sequences, the inverted repetitive sequences, forms hairpin-like structures in denatured DNA by the folding back of a single linear chain. The reassociation process of these sequences is unimolecular and the rate is extremely fast. Inverted repetitive sequences comprise 6% of the total human genome. They appear to be heterogeneous in length with an overall average length of 190 nucleotides. The inverted sequences are represented in almost all families of repetition frequencies, highly repetitive as well as very few copies per genome. They are not localized at unique sites on metaphase chromosomes. It is estimated that there are approximately 2 X 10(6) inverted repeats per haploid human genome. The biological function of this class of sequences is unknown.     

Interest of gene amplification by PCR for the diagnosis of Mycoplasma pneumoniae infections in the child

The relationship between preoperative serum carcinoembryonic antigen (CEA), CA 19-9 and alpha-fetoprotein (AFP) levels and their clinicopathological features were evaluated in gastric cancer patients. The positive rates of CEA, CA 19-9 and AFP were 24.8, 27.6 and 12.7%, respectively. Gastric cancer with deeper tumor invasion was significantly more common among patients positive for these tumor markers. Patients with positive CEA or CA 19-9 values had a significantly high risk of lymph node metastases (p = 0.045 and p = 0.002, respectively). Synchronous liver metastases was more commonly found in patients with a positive CA 19-9 value. A significant difference (p < 0.001) in survival rate was found between patients with positive CA 19-9 values and those with negative values. CA 19-9 is useful for the prognosis of gastric cancer patients, whereas CEA, although unsuitable for prognosis, contributes to the prediction of cancer invasion.     

Diagnosis of Mycoplasma pneumoniae infection by specific IgM antibodies using a new capture-enzyme-immunoassay

A total of 1226 sera from 1055 patients with respiratory tract infections were tested. IgM antibodies were detected by an antibody-capture enzyme-immunoassay (Mp TEST, Diatech Diagnostica Ltd, Israel). Acute infection with IgM antibodies to Mycoplasma pneumoniae was detected in 211 patients. Presence of IgM was closely associated to some or all pneumonia-related symptoms. Eighty-one of IgM-positive patients treated with tetracycline or erythromycin responded positively. Of the 211 patients, 63 (30%) had low levels and 23 (11%) had moderate levels of IgM antibodies already in the first serum sample. In these 86 patients (41%) the complement fixation (CF) test was negative or very low positive. Thus in these cases, the CF test would have missed the early diagnosis in the first serum samples.     

Systematic analysis of coding and noncoding DNA sequences using methods of statistical linguistics

We compare the statistical properties of coding and noncoding regions in eukaryotic and viral DNA sequences by adapting two tests developed for the analysis of natural languages and symbolic sequences. The data set comprises all 30 sequences of length above 50 000 base pairs in GenBank Release No. 81.0, as well as the recently published sequences of C. elegans chromosome III (2.2 Mbp) and yeast chromosome XI (661 Kbp). We find that for the three chromosomes we studied the statistical properties of noncoding regions appear to be closer to those observed in natural languages than those of coding regions. In particular, (i) a n-tuple Zipf analysis of noncoding regions reveals a regime close to power-law behavior while the coding regions show logarithmic behavior over a wide interval, while (ii) an n-gram entropy measurement shows that the noncoding regions have a lower n-gram entropy (and hence a larger "n-gram redundancy") than the coding regions. In contrast to the three chromosomes, we find that for vertebrates such as primates and rodents and for viral DNA, the difference between the statistical properties of coding and noncoding regions is not pronounced and therefore the results of the analyses of the investigated sequences are less conclusive. After noting the intrinsic limitations of the n-gram redundancy analysis, we also briefly discuss the failure of the zeroth- and first-order Markovian models or simple nucleotide repeats to account fully for these "linguistic" features of DNA. Finally, we emphasize that our results by no means prove the existence of a "language" in noncoding DNA.     

Endogenous retroviral sequences present in the human genome

The common carotid arteries of normal adult rats were investigated electron-microscopically after tannic acid fixation. This fixation technique yields a better demonstrability of the structures of the connective tissue, the basal laminae and the surface coat of the cell membrane. The common carotid artery represents a vessel of the elastic type. The intima consists of an endothelium and a narrow gap of connective tissue (0.1-1 micron) which contains single collagenous fibrils and small elastic structures. This space is only occasionally as wide as 3 microns, especially beneath gaps of the internal elastic membrane. In these areas, single cells and structures of densely packed filaments are additionally observed which can neither be attributed to collagenous fibrils nor to elastic fibres. The intima is demarcated from the outside by an internal elastic membrane (1 micron) which shows a number of gaps. The media exhibits 3 to 4 elastic membranes without gaps. Smooth muscle cells of the contractile type stretch in an oblique direction between these membranes, i.e. they are not arranged in a circular or spiral manner. Most of their process-rich ends are inserted directly into the elastic material and not via a basal lamina. Processes from these smooth muscle cells, collagenous fibrils and elastic fibres are seen in the intercellular spaces. The muscle cells are occasionally interlinked by gap junctions. The basal lamina does not surround the muscle cells continuously. The adventitia contains bundles of collagenous fibrils, fibrocytes, a few small vessels and nerves with a perineuronal envelope. Nerves could not be demonstrated in the media. The oblique course of the smooth muscle cells and the insertion into the elastic membranes indicate that these cells do not predominantly contribute to changes in the width of the lumen but also serve the stabilisation and resetting of the elastic membranes. Contraction is probably induced by an opening of stretch-dependent Ca2+ channels. Due to the interlinkage with gap junctions, the muscle cells of one layer respond as a functional unit. Our findings provide a morphological basis for elucidating commonly encountered changes, such as smooth muscle migration through a normally interrupted inner elastic lamina.     

The organization of DNA sequences in the mouse genome

Analysis of the organization of nucleotide sequences in mouse genome is carried out on total DNA at different fragment size, reannealed to intermediate value of Cot, by Ag+--Cs2SO4 density gradient centrifugation.--According to nuclease S-1 resistance and kinetic renaturation curves mouse genome appears to be made up of non-repetitive DNA (76% of total DNA), middle repetitive DNA (average repetition frequency 2X10(4) copies, 15% of total DNA), highly repetitive DNA (8% of total DNA) and fold-back DNA (renatured density 1.701 g/ml, 1% of total DNA).--Non-repetitive sequences are intercalated with short middle repetitive sequences. One third of non-repetitive sequences is longer than 4500 nucleotides, another third is long between 1800 and 4500 nucleotides, and the remainder is shorter than 1800 nucleotides.--Middle repetitive sequences are transcribed in vivo. The majority of the transcribed repeated sequences appears to be not linked to the bulk of non-repeated sequences at a DNA size of 1800 nucleotides.--The organization of mouse genome analyzed by Ag+--Cs2SO4 density gradient of reannealed DNA appears to be substantially different than that previously observed in human genome using the same technique.     

Seroepidemiology of Mycoplasma pneumoniae infections in Iceland 1987-96

Friedreich's ataxia is an inherited disorder of the nervous system, requiring special care during anaesthesia, because of increased sensitivity to muscle relaxants. We report a case of Friedreich's ataxia in a 31-year-old woman, anaesthetised on two occasions, for tendinoplasty and pes cavus repair. Atracurium was used for neuromuscular blockade and monitored by a train-of-four twitch technique. The patient's response was normal. She returned to adequate spontaneous breathing within 20 min of the last dose of the muscle relaxant without need for anticholinesterase administration. When neuromuscular function is monitored, normal doses of muscle relaxant can safely be used in these patients.     

Electron microscopy of Mycoplasma pneumoniae microcolonies grown on solid surfaces

Mycoplasma pneumoniae sprain CL-8 was studied by using various surfaces for adherence and growth. Cells grown on Epon 812, Formvar, carbon, and glass were of similar morphology. Thin Epon pieces were good material for culturing the organisms and examining thin-sectioned microcolonies by transmission electron microscopy.     

A longitudinal study of Mycoplasma pneumoniae: infections in Navy recruits by isolation and seroepidemiology

A longitudinal serologic study of M. pneumoniae infections in two recruit training centers with grossly different climates showed that the frequency of this infection peaks in the late summer-early fall and to a lesser degree in the spring at both training centers. Seroconversions (four-fold or greater increase in titer) were as high as 45-57% in some recruit companies during the late summer peak. M. pneumoniae isolations from men admitted to the dispensary or hospital with pneumonia during the major peak season were as high as 32%. While M. pneumoniae infections are not generally life-threatening, the high incidence of infections observed in personnel during recruit training is of major concern and warrants consideration of effective control measures.     

A case of hypoxemic acute bronchiolitis presenting with diffuse nodular shadows caused by Mycoplasma pneumoniae

A fifty year-old female who had previously been well presented with a productive cough and a high fever. Her initial chest X-ray film showed no abnormal lung shadows. Despite partial improvement of the fever and the serum level of acute phase reactant (CRP) in response to intravenous administration of piperacillin, she complained of increasing severity of cough and dyspnea. Follow-up chest X-ray films taken five days after therapy with piperacillin showed diffuse nodular shadows in the mid-to-lower lung fields bilaterally. Chest CT scan disclosed diffuse miliary nodules at the lung periphery and thickening of bronchovascular markings. Chest auscultation revealed late inspiratory coarse crackles and expiratory wheezing, and the patient's arterial oxygen tension was 61 mmHg. Suspected of suffering from primary atypical pneumonia, she was started on therapy with intravenous minocyclin (200 mg/day), two days after treatment her symptoms began improving significantly. Anti-mycoplasma antibody was found to be x 1280, and cold hemoagglutinin x 1024, establishing the diagnosis of Mycoplasma pneumoniae infection. The patient's condition completely recovered following a one week treatment with minocyclin. We concluded that her respiratory infection was caused by piperacillin-sensitive mico-organism, and also Mycoplasma pneumoniae which brought about hypoxic acute bronchiolitis to the patient.     

Diagnosis of Mycoplasma pneumoniae pneumonia in pediatric patients by polymerase chain reaction (PCR)

Polymerase chain reaction (PCR) testing was performed on respiratory tract specimens obtained by throat swab in 21 children admitted to the hospital with suspected Mycoplasma pneumoniae pneumonia. Of 13 patients with a clinical condition compatible with mycoplasma infection and an immunological response to M. pneumoniae, 11 were positive by PCR. Eight patients were negative by serology and/or had a clinical condition not compatible with mycoplasma infection, and all were negative by PCR. The antibody response to M. pneumoniae was delayed for a week or more in 3 (23%) of the 13 patients with documented mycoplasma infection. These results suggest that PCR performed on a respiratory tract specimen obtained by a throat swab may be useful in the initial evaluation of children with suspected M. pneumoniae pneumonia, especially in patients in whom the serological response is delayed.