Iranian Journal of Biotechnology
Volume:3 Issue: 4, Autumn 2005

The genomic and cDNA clones encoding cellobiohydrolase II (cbhII) have been isolated and sequenced from a native Iranian isolate of Trichoderma parceramosum, a high cellulolytic enzymes producer isolate. This represents the first report of cbhII gene from this organism. Comparison of genomic and cDNA sequences indicates this gene contains three short introns and also an open reading frame coding for a protein of 470 amino acids. The deduced amino acid sequence includes a putative signal peptide, cellulose binding domain, linker region, and catalytic domain. Homology between this sequence and other reported Trichoderma CBHII proteins and also structural prediction of this enzyme are discussed. The coding sequence of cbhII gene was cloned in pPIC9 expression vector and expressed in Pichia pastoris GS115. The expression was confirmed by Northern dot blot, RT-PCR and enzyme activity staining

Two Arabidopsis thaliana genes, psr9.2 and psr9.4 appeared to be highly similar to a phosphate-starved induced gene, psr9, isolated from Brassica nigra suspension cells. Sequence analysis classified the encoded polypeptides as members of leucine-rich repeat (LRR) proteins superfamily. The sequence of psr9 proteins comprise a unique N-terminal region encompassing a coiled-coil structure proceeding eleven LRRs along the C-terminal. Expression pattern analysis showed the responsiveness of these genes to various environmental conditions. Although both psr9 genes, psr9.2 and psr9.4, are expressed throughout the plant, the expression of psr9.2 was higher in the root whereas psr9.4 expression was prominent in the shoot. The expression levels were increased proportional to the duration of phosphate deprivation treatment. Plants exposed to cold temperature expressed both genes at high levels in both roots and shoots. In contrast, heat shock increased the expression levels of both genes in the shoots while reducing it in roots. High-salt treatment upregulated the expression of psr9 genes only in the roots. These data may suggest distinct roles for psr9 genes during plant response to various environmental conditions.Keywords: psr9; Cold stress; High salt stress; Phosphate starvation

In this study, the genetic diversity of 12 Iranian Damask rose (Rosa damascena Mill.) genotypes was studied using amplified fragment length polymorphism (AFLP) markers. Twelve AFLP primer combinations generated 483 polymorphic bands and showed extreme variability and genetic complexity among the studied genotypes. The AFLP analysis revealed a specific amplified fragment for the genotypes collected from the Meymand region. Despite the geographical distance between Kashan and Kazeroon regions, the results of cluster analysis showed that the genotypes collected from these districts regions were genetically related. The low genetic similarities were observed among the genotypes belonging to Tabriz, Meymand and Kashan regions suggesting that there was relatively less geographical relocation of the genotypes between these regions. Hence, for breeding of the Damask roses taken in this study, it is necessary to conjunct the molecular data with agronomic characters.

A study was undertaken to evaluate the genetic variation in the 10th generation of a breeder flock of native breed selected for high egg and meat production in native fowls breeding station, Mazandaran, Iran. Venous blood samples were collected from 100 birds of both sexes. The RAPD-PCR technique was applied to generate a DNA fingerprint of individuals. Initially, a total of 20 ten-nucleotide arbitrary primers were used but 14 of 20 primers revealed a pattern with scorable amplified bands. From a total number of 140 scored bands 63 (45%) and 77 (55%) were described as polymorphic and monophormic respectively. The average number of bands per primer varied from 4 to 16 and with sizes varying from 200 to 2100 bp in length. The estimated band sharing frequency varied from 0.79 to 0.96 between individuals. The average genetic similarity and genetic variance between individuals within the population were 0.89 and 0.11, respectively. The existence of high levels of polymorphism after 10th generation of selection may indicate the accuracy of used the selection program and also the large enough effective population size in this breeding flock. It could be concluded that RAPD markers are effective in detecting genetic similarities and genetic variances among individuals in poultry breeder flocks.Keywords: RAPD markers; Genetic variability; Native fowl

Production of single cell protein (SCP) from natural gas in a one liter bubble column reactor and optimization of the process parameters were investigated. The medium specifications, nitrogen sources, initial inoculum volume, and inlet ratio of gas to air were considered as process parameters to be optimized. The optimum condition for highest biomass production in which the maximum quantity of protein was obtained, were the use of a certain carbon-less salt broth utilizable with methane (named as Methane Salt Broth/MSB) and sodium nitrate as medium and nitrogen source respectively. Also, 7%(v/v) inoculation size, and an inlet gas mixture of 60/40 natural gas/air were determined as the effective inoculation and appropriate volume configuration of inlet gases. Protein production in optimum condition was 69.3%(w/w) of biomass in dry basis which its structural amino acids can be in comparable with other nutrient sources. An average amount of 10 g RNA out of 100 g of cellular protein [or 6%(w/w) RNA in whole biomass] was extracted from the biomass which is extremely near to the possible minimum of RNA distribution among bacteria. Heat shock treatment was applied for reducing the RNA in the biomass bulk. Heat Treatment at 60 to 65C for 10 to 20 min provided the best RNA reduction results (around 1 gram in 100 grams of protein). Regarding the structural amino acids and RNA content, the properties of single cell protein resulted in this experimental work, were in a frame which it could be consumed safely.

Many kinds of mutations in mitochondrial (mt) DNA have been reported to be related to the development of Diabetes Mellitus (DM), this type of diabetes has also been shown to be influenced by other genetic factors and/or environmental factors. Among them, tRNALeu(UUR) and its adjacent mtDNA NADH dehydrogenase subunit 1(ND1) region within the mt genome are linked to high susceptibility to DM. A point mutation at 3243 base pair (bp) in the mt tRNA Leu(UUR) is commonly referred to as a syndrome of mitochondrial myopathy, Encephalopathy, Lactic acidosis, and Stroke-like episodes (MELAS). In the current study, we have assessed the frequency of the A3243G in Iranian diabetic type 2 patients. DNA was obtained from peripheral leukocytes of 154 patients with diabetes Mellitus type2 (l50 with type 2 and 4 with gestational diabetes) and 40 control subjects. Insulin concentration from patients blood was measured using Radioimmunoassay procedure. Patients showed fasting blood sugar (FBS) between 150-230 mg/dl, body mass index (BMI) between 19-32 Kg/m2 and insulin concentration 0.9-2.35 mg/ml. PCR-RFLP, single strand conformation polymorphism (SSCP) and sequencing methods were used to detect the A3243G or other mutations in the mitochondrial tRNALeu (UUR) gene. A3243G mutation was not detected in patients. SSCP results showed a new pattern of PCR product in 6 patients. The C3316T transition mutation in the ND1 mitochondrial gene was confirmed in selected samples (n=6) by sequencing. No differences were observed between the two groups for C3316T and A3243G mutations (P=0.348). The mt C3316T mutation did not have any effect on the clinical finding of type 2 diabetes carrying this mutation. These data together with clinical characteristics of the patients may suggest that the mt C3316T mutation might be a polymorphism in the Iranian population.Keywords: Type 2 Diabetes; Mitochondria; A3243G mutation; C3316T mutation

Alpha 1-antitrypsin (AAT) or alpha 1-protease inhibitor (PI) is the principal inhibitor of proteolytic enzyme in serum. Its phenotypic variability has been reported to be associated with liver, lung diseases and rheumatoid arthritis in humans. There is much documentation about high risk phenotypes of PI in some regions of the world, however, there are no reliable reports on these phenotypes and genotypes and their related diseases in Iranian population. The aim of this study was to determine PI phenotypes and genotypes in Iranian patients suffering from PI deficiency. For this purpose, whole blood samples from 307 patients suspected of diseases related to PI deficiency, and 156 healthy persons were examined. PI phenotypes and genotypes were determined by isoelectric focusing (IEF) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), respectively. Allele frequencies from patients and normal subjects were compared. For reliability, a family study of the patients was also carried out. The PI phenotype frequencies of all six possible combinations of M, S and Z haplotypes in patients were: MM, 77.20%; MS, 6.18%; MZ, 7.17%; SS, 3.91%; ZZ, 4.56%; SZ, 0.98% and in normal subjects were: MM, 78.20%; MS, 5.76%; MZ, 15.38%; SS, 0.64%; 0% for ZZ and SZ. Analysis of data showed that there was a significant difference between patients (with liver, lung diseases and rheumatoid arthritis) and control subjects (p < 0.05). In Conclusion, the allelic frequencies of S and Z in the patient group were 7.49% and 8.63%, while in the normal subjects were 5.13% and 4.17%, respectively. This is the first report of the prevalence of high risk alleles (Z and S) in patients suspected of PI deficiency and related diseases in Iran.Keywords: Alpha 1-antitrypsin deficiency; Phenotypes; Genotypes; IEF; PCR-RFLP

Mutations in the connexin 26 (Cx26) gene at the DFNB1 locus on chromosome 13q12 are associated with autosomal recessive non-syndromic hearing loss (ARNSHL). There are many known mutations in this gene that cause hearing loss. A single frameshift, at position 35 (35delG) accounts for 50% of mutations in the Caucasian population with carrier frequencies of 1.5-2.5%. In this study we investigated the prevalence of Cx26 gene mutations by directly sequencing the coding exon of this gene belonging to ARNSHL individuals from 53 families in Qom and Markazi provinces of Iran. Seven different Cx26 variants were identified. Five Cx26 mutations including 35delG, 233delC, 176del16, W24X, L90P were found in 10 of 53 families (18.87%). One polymorphism V153I was also found. One variant A171T with unknown effects was also detected. Six of the 53 families were observed to have GJB2 mutations in both alleles (11.32%). The most common mutation was 35delG. Three out of 10 families (30%) with GJB2 variants contained 35delG mutation in both alleles and the frequency of 35delG allele was 0.50 among 10 out of 53 families. Also screening for the 342-kb GJB6 deletion mutant did not reveal any large deletion among families studied. Thus, in the two provinces, contribution of GJB2 (Gap Junction Protein Beta 2) mutations to familial deafness appears to be less significant. This necessitates further assessment of the other known genes regions as well as a search for new genetic factors in hereditary deafness in the Iranian population.