Construction of Mycoplasma hyopneumoniae P97 Null Mutants

Mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia, a world-wide problem in the pig industry. This disease is characterized by a dry, non-productive cough, labored breathing, and pneumonia. Despite years of research, vaccines are marginally effective, and none fully protect pigs in a production environment. A better understanding of the host-pathogen interactions of the M. hyopneumoniae-pig disease, which are complex and involve both host and pathogen components, is required. Among the surface proteins involved in virulence are members of two gene families called P97 and P102. These proteins are the adhesins directing attachment of the organism to the swine respiratory epithelium. P97 is the major ciliary binding adhesin and has been studied extensively. Monoclonal antibodies that block its binding to swine cilia have contributed extensively to its characterization. In this study we use recombination to construct null mutants of P97 in M. hyopneumoniae and characterize the resulting mutants in terms of loss of protein by immunoblot using monoclonal antibodies, ability to bind purified swine cilia, and adherence to PK15 cells. Various approaches to recombination with this fastidious mycoplasma were tested including intact plasmid DNA, single-stranded DNA, and linear DNA with and without a heterologous RecA protein. Our results indicate that recombination can be used to generate site-specific mutants in M. hyopneumoniae. P97 mutants are deficient in cilia binding and PK15 cell adherence, and lack the characteristic banding pattern seen in immunoblots developed with the anti-P97 monoclonal antibody.

Using Random Amplified Polymorphic DNA (RAPD) analysis to investigation of genetic diversity, and relationships among a set of clinical Aspergillus fumigatus isolates

This study is an attempt to determine the genetic diversity and relationships among fourteen local isolate isolated from patients with Aspergillosis (Aspergillus fumigatus) by using the Random Amplified Polymorphic DNA (RAPD) technique. Twelve universal primers used in this study produced 94 bands across fourteen isolates. Of these bands, 67 bands or 71.2% were polymorphic. The size of the amplified bands ranged between 100-2000 bp. The genetic polymorphism value of each primer was determined and ranged between 33-100%. In terms of unique banding patterns, determine the finger print for six isolates the most characteristic banding pattern was for the (AFU1, AFU2, AFU3, AFU4, AFU8 and AFU14) with primer (OP F-16 , OP I-06, OP F-16, OP X-01, OP X-01and OP A-06). Genetic distances ranged from 0.12419 to 0.64404 among A. fumigatus isolates. Cluster analyses were performed to construct a dendrogram among studied A. fumigatus isolates. The cluster analysis places most of the A.fumigatus isolates isolated from patient come from yhe same area into a close relation (subcluster) showing a high level of genetic relatedness and were distinct from isolates from another area (the other subcluster). Interestingly, a number of isolates originating from the same sources did form well defined groups, indicating association between the RAPD patterns and the geographic origin of the isolates. The information generated from this study can be used in the future for controlling of Aspergillosis programs.

Vertical tectonic motion in Andaman Islands detected by multi-temporal satellite radar images

On 26 December 2004 one of the largest earthquakes ever recorded by modern seismology (Mw>9.0) occurred in Sumatra-Andaman region. About 1300km of seafloor were ruptured extending from northern Sumatra to Andaman Island complex. As instrumental data are sparse and in most of the cases field observations difficult to undertake, the role of remote sensing may be beneficial in providing information about the pattern of deformation in space and time. In the present study ENVISAT ASAR data are acquired and processed to generate a single multitemporal SAR image in order to detect vertical motion along Andaman Island. According to our analysis it seems that the northern parts of the island are generally uplifted. Though its north-western coasts show significant marL· of uplift, such indications become less evident along the western coasts of Middle Andaman. At some locations at the south-eastern margins of the island marks of downlift are recognized. Characteristic banding observed in the multi-temporal image could be attributed either to post-seismic relaxation, either to tidal phenomena. Although qualitative in nature, as just the sign of elevation change can be detected, results of such analysis could be used as constrains in deformation modelling research.

Strain differentiation of Trichophyton rubrum by randomly amplified polymorphic DNA and analysis of rDNA nontranscribed spacer

Trichophyton rubrum is the most common pathogen causing dermatophytosis. Molecular strain-typing methods have recently been developed to tackle epidemiological questions and the problem of relapse following treatment. A total of 67 strains of T. rubrum were screened for genetic variation by randomly amplified polymorphic DNA (RAPD) analysis, with two primers, 5′-d[GGTGCGGGAA]-3′ and 5′-d[CCCGTCAGCA]-3′, as well as by subrepeat element analysis of the nontranscribed spacer of rDNA, using the repetitive subelements TRS-1 and TRS-2. A total of 12 individual patterns were recognized with the first primer and 11 with the second. Phylogenetic analysis of the RAPD products showed a high degree of similarity (>90 %) among the epidemiologically related clinical isolates, while the other strains possessed 60 % similarity. Specific amplification of TRS-1 produced three strain-characteristic banding patterns (PCR types); simple patterns representing one copy of TRS-1 and two copies of TRS-2 accounted for around 85 % of all isolates. It is concluded that molecular analysis has important implications for epidemiological studies, and RAPD analysis is especially suitable for molecular typing in T. rubrum.

Strain Identification of Trichophyton rubrum by Specific Amplification of Subrepeat Elements in the Ribosomal DNA Nontranscribed Spacer

Trichophyton rubrum is the commonest cause of dermatophytosis of skin and nail tissue. Molecular characterization of the T. rubrum ribosomal DNA nontranscribed-spacer region revealed two novel tandemly repetitive subelements (TRSs): TRS-1, containing a 27-bp palindromic sequence, and TRS-2. Specific amplification of TRS-1 produced strain-characteristic banding patterns (PCR types), with 21 TRS-1 PCR types recognized from 101 clinical isolates. Four simple patterns representing 1 to 4 copies of TRS-1 accounted for 75 (75%) of all 101 strains, whereas more complex patterns were observed for 21 (20%) of the 101 isolates. The copy number of TRS-2 was 0 to 3 repeats per cistron, with a majority of isolates having two copies of this element. Eleven isolates were polymorphic for TRS-2, and in combination, 23 separate PCR types were recognized by amplification of both TRS-1 and TRS-2. The PCR patterns from both elements were stable and reproducible. Elements with homology to TRS-1 were present in three phylogenetically related species,Trichophyton violaceum, Trichophyton gourvilii, and Trichophyton soudanense, but these elements were not identified in other dermatophyte taxa. There was no clear correlation of PCR type with specimen (skin or nail tissue), but certain PCR types appeared to show a bias in geographic distribution. This new method of typing T. rubrum will enable important questions about pathogenesis and epidemiology of this fungus to be addressed.

Banding of rye (Secale cereale) chromosomes using the restriction enzymes AluI, DraI, HpaII, and MspI

Mitotic metaphase chromosomes of the rye inbred line L 301, which belongs to the Sortiment of the University of Hohenheim, were treated in situ with the restriction enzymes AluI (recognition sequence: 5′-AC/GT-3′), DraI (recognition sequence: 5′-TTT/AAA-3′), and the isoschizomeres HpaII and MspI (recognition sequence: 5′-C/CGG-3′) and stained with Giemsa. The chromosomes indicated similar banding patterns in comparison with the conventional Giemsa-C-banding. However, we have found in rye chromosomes after restrictase treatment that the telomeric bands were reduced in extension. In a lower degree the centromeric bands of individual chromosomes could be absent in dependence of the used restriction enzymes. The number of the intercalary bands were also reduced. Nevertheless, the tested restriction enzymes produced characteristic banding patterns of the rye genome. This uncomplicated banding technique is suited for a very quick banding method of karyotype analysis especially to obtain a first survey of the band patterns on the rye chromosomes.Key words: Secale cereale L., chromosome band pattern, in situ digestion, restriction endonuclease, restriction banding.

High-mobility group protein HMG-I localizes to G/Q- and C-bands of human and mouse chromosomes.

Mammalian metaphase chromosomes can be identified by their characteristic banding pattern when stained with Giemsa dye after brief proteolytic digestion. The resulting G-bands are known to contain regions of DNA enriched in A/T residues and to be the principal location for the L1 (or Kpn 1) family of long interspersed repetitive sequences in human chromosomes. Here we report that antibodies raised against a highly purified and biochemically well characterized nonhistone "High-Mobility Group" protein, HMG-I, specifically localize this protein to the G-bands in mammalian metaphase chromosomes. In some preparations in which chromosomes are highly condensed, HMG-I appears to be located at the centromere and/or telomere regions of mammalian chromosomes as well. To our knowledge, this is the first well-characterized mammalian protein that localizes primarily to G-band regions of chromosomes.

Interconversion of structural and contractile actin gels by insertion of myosin during assembly.

Extracts of the soluble cytoplasmic proteins of the sea urchin egg form gels of different composition and properties depending on the temperature used to induce actin polymerization. At temperatures that inactivate myosin, a gel composed of actin, fascin, and a 220,000-mol-wt protein is formed. Fascin binds actin into highly organized units with a characteristic banding pattern, and these actin-fascin units are the structural core of the sea urchin microvilli formed after fertilization and of the urchin coelomocyte filopods. Under milder conditions a more complex myosin-containing gel is formed, which contracts to a small fraction of its original volume within an hour after formation. What has been called "structural" gel can be assembled by combining actin, fascin, and the 220,000-mol-wt protein in 50-100 mM KCl; the aim of the experiments reported here was to determine whether myosin could be included during assembly, thereby interconverting structural and contractile gel. This approach is limited by the aggregation of sea urchin myosin at the low salt concentrations utilized in gel assembly. A method has been devised for the sequential combination of these components under controlled KCl and ATP concentrations that allows the formation of a gel containing dispersed myosin at a final concentration of 60-100 mM KCl. These gels are stable at low (approximately 10 micron) ATP concentrations, but contract to a small volume in the presence of higher (approximately 100 micron) ATP. Contraction can be controlled by forming a stable gel at low ATP and then overlaying it with a solution containing sufficient ATP to induce contraction. This system may provide a useful model for the study of the interrelations between cytoplasmic structure and motility.

A SUGGESTION FOR THE NOMENCLATURE OF THE FLUORESCENT BANDING PATTERNS IN HUMAN METAPHASE CHROMOSOMES

With the application of fluorescent technique, it is now possible to recognize characteristic banding patterns in human chromosomes. A simple nomenclature for the bands is suggested according to their visual identification.