مجله بیوتکنولوژی ایران
سال:1382 | دوره: | شماره: |تعداد مقالات:8

Mitochondria contain their own DNA (mtDNA), which codes for 13 proteins (all subunits of the respiratory chain complexes), 22 tRNAs and 2 rRNAs. Several mtDNA point mutations as well as deletions have been shown to be causative in well-defined mitochondrial disorders. A mixture of mutated and wild type mtDNA (heteroplasmy) is found in most of these disorders. Inheritance of mtDNA is maternal, and mothers with heteroplasmic mtDNA transmit different proportions of normal and mutated mtDNA to the children. Mitochondrial tRNA genes have a central role in mitochondrial gene expression at the level of transcription, RNA processing and protein synthesis and they appear to be the mitochondrial genes most frequently affected by mutations causing diseases in man.

Chronic Myeloid Leukemia (CML) is a hematopoietic malignancy characterized by the presence of Philadelphia (Ph1) chromosome that results from balanced reciprocal translocation between chromosomes 9 and 22 leading in the formation of bcr/abl fusion gene. The present study was conducted to evaluate cytogenetic and molecular abnormalities in CML patients at presentation and during the course of therapy. Cytogenetic analysis was carried out in bone marrow samples of 165 suspected patients of CML using standard protocols. Sequential cytogenetic analysis was also done in 55 CML patients (50 on IFN-α 2b therapy and 5 on Hydroxyurea) up to variable period of 3 years. Fluorescence In Situ Hybridization (FISH) using specific probes for bcr and abl genes was carried out in cases where conventional cytogenetics was not informative, in cases that were Ph-negative at presentation and in cases with complete or major cytogenetic response (<34% Ph+cells). Of the 165 CML patients, 157 patients (95%) were Ph-positive while 8 patients (5%) were Ph-negative. Cytogenetic abnormalities other than the standard Ph1 translocation were observed in 2 Ph+ patients and 2 Ph-negative patients. Sequential cytogenetic analysis revealed varied degrees of cytogenetic response in 35 patients on IFN-α 2b therapy. Complete cytogenetic response was observed in 8 patients (16%) on IFN-α 2b therapy. However, FISH analysis could detect bcr/abl fusion gene in some of these cases. This finding highlights the sensitivity of this technique in detecting residual disease even in patients with complete cytogenetic remission. The other patients in complete cytogenetic remission did not have evidence of bcr/abl fusion. FISH analysis also revealed bcr/abl fusion signals in Ph-negative cases (with no cytogenetic abnormality) indicating a masked translocation or a sub-microscopic rearrangement. Thus, the results of the present study show that analysis of cancer patients on therapy at the molecular level has tremendous importance for management of CML patients. Further, with the use of highly sensitive FISH technique, results can be obtained within 24 hours thereby, aiding rapid diagnosis and management of these patients. This efficient and highly sensitive molecular cytogenetic technique can also be done on interphase cells and poorly spread metaphases thereby, overcoming the difficulty of conventional chromosomal analysis especially in patients on therapy

Denaturing High-Performance Liquid Chromatography (DHPLC) is a recently developed technique for the detection of single nucleotide polymorphisms (SNPs) and mutations. It involves the comparison between two or more DNAs as a mixture of denatured and reannealed PCR products. The methodology is based on the principle of reversed phase liquid chromatography and uses a unique DNA separation matrix. The exquisite sensitivity of the technique is determined by adjusting the oven temperature. Elution of DNA fragments is dependant on the chain length and sequence and can be predicted by computation. Under partially denaturing conditions heteroduplices formed upon mixing, denaturing, and reannealing of two, or more, chromosomes that differ in sequence, are retained less than their corresponding homodupulices and sequence variation is recognized by the appearance of two, or more, peaks in the chromatographs. Numerous SNPs have been identified on the non-recombinant portion of the Y chromosome by using this technique. To investigate the DNA variation within Pakistan 15 Y-SNPs, an Alu insertion, a LINE1 insertion and the 12f2 deletion, mapping on the non-recombining portion of the human Y chromosome, were typed in 834 Pakistani males. The combination of these biallelic markers identified 11 stable Y chromosomal lineages or Y ‘haplogroups’ in the Pakistani population. Haplogroup frequencies were generally similar tothose in neighboring geographical areas and indicate that there was a common pool of Y lineages within Pakistan that are predominantly from West and Central Asia.

The standard method for immunoscreening of a cDNA expression library is time-consuming because of the production of a large proportion of false positives during the first and second round of screening. This problem is more important when a sensitive chemiluminescence detection system is used. Due to the high sensitivity of the detection system, there is a need to avoid false positives which occur when the antibody reacts non-specifically. False positives are generally eliminated through absorption of the antibody with the host bacteria and by eliminating any clones, which react with antibodies present in normal sera. Here we present a method of obtaining almost identical bacteriophage plates by culturing phage in parallel, and show that this technique produces positive plaques in duplicate and eliminates false positives. Using this method, we successfully screened a human fetal spinal cord lambda gt11 cDNA library using purified immunoglobulin G (IgG) from patients with multiple sclerosis (MS) and Guillain – Barre syndrome (GBS).

The Rhodococcus FMF (R. FMF) native bacterium was isolated from soil contaminated with oil in Tabriz refinery. This bacterium carries three genes sox (dszA, B, C) on its genomic DNA. Preliminary studies have proved that R. FMF strain possess desulfurization activity. In this work soxA and B genes were amplified by PCR, after designing a pair of suitable primers. Analysis of PCR products on agarose gel electrophoresis showed a sharp band in the 2.46 kb region, belonging to soxA and B genes. In addition, dot blotting using the sox4 probe, confirmed the presence of sox operon in the PCR product. The standard Gibbs test was designed for desulfurization activity assay in which production of 2HBP (µM) in four bacterial strains [R. FMF, P. aeruginosa (pESOX4), E.coli DH5 α(pESOX3) and E. coli CC118λpir (pESOX4)] was recognized and compared. After 41h of dibenzothiophene (DBT) addition, Pseudomonas aeruginosa (pESOX4) produced 4.8 µM 2HBP and the local R. FMF produced 3.8 µM of DBT. Comparison of 2HBP production in the standard strain of P. aeruginosa (pESOX4) with the isolated R. FMF strain from Tabriz refinery revealed that the later is equally capable of desulfurizing DBT.

The mechanism of plasmid mediated silver (Ag) resistance was investigated in Acinetobacter baumannii BL54. The intracellular accumulation of Ag in both original strain BL54 and Escherichia coli K12 transconjugant containing plasmid pUPI276 began immediately and reached a maximum within 60 minutes. This initial accumulation was followed by net loss of Ag which reached a maximum within 180 min. Pre-treatment of cells with 0.5 mM 2,4 dinitrophenol (DNP); 20 mM N, N-dicyclohexylcarbodiimide (DCCD); 3% toluene; 25 mg/ml cefotaxime and polymyxin-B resulted in considerable decrease in the accumulation process. Ags plasmid less cured derivative (BL54.1) also accumulated silver but only onefourth the amount compared to the resistant strain BL54. The intracellular accumulated silver is detoxified by binding to a cysteine rich metal binding protein. The purified Ag-binding protein exhibited maximum absorption at 280/215 nm. From the above data it could be concluded that the intracellular detoxification of silver in A. baumannii BL54 is achieved through binding to a cysteine rich metalloprotein.

Blast, Pyricularia grisea (Cooke) Sacc., is one of the most destructive diseases of rice worldwide and can result in significant reductions in yield. The use of resistant cultivars is the most economical and effective way of controlling rice blast. A variety of DNA markers, including plant defense-related candidate gene markers are available for genetic characterization and molecular analysis of rice. A set of 161 recombinant inbred lines, RILs, from a cross between Nemat, an improved and high yielding cultivar, and Anbarboo, a traditional and aromatic rice, was used to identify defense-related candidate gene, RFLP and SSR markers linked to components of resistance to blast, i.e. infection type, lesion density, the percent of diseased leaf area, and lesion size in rice. The RILs were tested using two single blast isolates in greenhouse, and field population of blast in blast nursery in International Rice Research Institute, Philippines, in 2000–2001. Of the 86 defense-related candidate gene, 153 RFLP, and SSR markers 26 defense-related candidate gene, 66 RFLP, and 85 SSR markers were polymorphic in two parental lines. Results showed that a defense gene, b8, a NBS-LRR originated from barley, closely linked to different components of resistance to blast. The defense genes of r5, r7, PrP2, and ERS from rice, maize, and Arabidopsis, respectively, have had minor effects on different components of resistance to blast. The RFLP markers, i.e. RZ536, RG351, RZ76, RZ397 on chromosomes 7, 11 and 12, and the SSR markers including RM224, RM179, and RM277 on chromosomes 11 and 12 were tightly linked to components of resistance to blast. The linked markers can now be used for resistance gene pyramiding and markerassisted selection in the breeding population. The results suggested the presence of race-specific resistance genes exhibiting strong differential pathogenhost interaction. We need to incorporate new sources of gene pool to make the genetic base broaden.

Polymerase chain reaction (PCR) is a rapid and simple technique with high sensitivity and specificity. In the recent years, PCR has been used for rapid detection of viral nucleic acids, such as Human cytomegalovirus (HCMV), whereas, PCR optimization is an important task to be done, especially before it’s diagnostic application. Annealing temperature, ion concentration (especially Mg2+ ion) and the cycling program and enhancer compounds are important optimization parameters. Peripheral blood leukocytes (PBLs) were isolated from samples collected from renal transplant recipients suffering from severe and symptomatic CMV disease. PBLs DNA was extracted and used for PCR. Annealing temperature and MgCl2 concentration and cycling condition were optimized. Dimethyl sulfoxide (DMSO) and gelatin were checked as enhancer components. The optimized condition obtained through this study was: 1x PCR buffer (20 mM Tris-HCl pH 8.6, 50 mM KCl), 2.5 mM MgCl2, 0.2 mM of each dNTPs, 0.25 µM of each primers, 0.25 unit/25 µl Taq DNA polymerase, 5% DMSO, 500 µg/ml gelatin and 50-150 ng template DNA in 25 µl final volume. PCR was performed as: 95°C 5 min (pre-denaturation), 94°C 50 sec, 58°C 1 min, 72°C 1 min for 35 cycles and 72°C 5 min (final extension). Using these conditions, it was shown that optimized PCR was five fold more sensitive than initial PCR; which can be used for diagnostic application of HCMV in renal transplant patients.