منوچهرتوسلی

The screening of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene groups is a quick way to show the potential for antibacterial agent production and novel drugs discovery. With regard to this matter, the presence and the phylogenetic relationship of these gene clusters were studied in three Streptomyces strains.

 Materials & Methods:

PCR amplification of PKS types I, PKS type II, and NRPS genes was performed using three degenerate primer sets in Streptomyces strains Iz8, F9 and F6, respectively. To separate the same-sized DNA fragments with different sequences, PCR products cloned to pTG19-T vector and then transformed to Escherichia coli TOP10. Polyacrylamide gel was used for the sequences variation. The purified plasmids were sequenced and phylogenetic trees were constructed using Bayesian inference, implemented in the MrBayes.

PKS I clone 3 and 8, PKS II clone 4 and NRPS clone 5 were deposited in the NCBI GenBank database. Phylogenetic tree from aligned nucletotide or amino acide sequences of PKS I and II contained several differential clades. PKS I clone 8 and PKS II clone 4 were one of these clades. This could raise the production possibility of new antimicrobial agents.

Construction of phylogenetic tree and determination of evolutionary relationships confirmed possibility of the presence of gene clusters with new compositions and therefore new products in strains Iz8 and F9.

The PIK3CA oncogene, which encodes p110α, is one of the most mutated genes in human cancers such as colorectal. A polymorphic GT dinucleotide repeat exists in intron 1 of the PIK3CA gene. Till this research, there were no study on polymorphism of PIK3CA gene microsatellites and their relationship with cancer risk. In the present study, we investigated GT repeat polymorphism in the intron 1 of this gene among patients with colorectal cancer and healthy individuals and evaluated the association between this polymorphism and the potential genetic susceptibility to the development of colorectal cancer.

Peripheral blood samples were collected from 103 patients with colorectal cancer and 100 healthy blood donors. After DNA extraction, GT dinucleotide region was amplified using polymerase chain reaction (PCR) technique and the number of GT repeats was determined via polyacrylamide gel electrophoresis.

Eight distinct alleles were identified in these subjects, ranging in size from 13 to 20 GT repeats. People with two alleles shorter than 17 GT repeat had a significantly higher risk of developing colorectal cancer (OR = 3.65, P = 9 × 10-6); in contrast, people with two alleles longer than 16 GT repeat were at a significantly lower risk of colorectal cancer (OR = 0.18, P = 3.5 × 10-6).

Our findings indicate significant relationship between the numbers of repetitive sequences in intron 1 of PIK3CA gene and the risk of colorectal cancer.

Colorectal cancer is the third leading cause of cancer deaths in men and the fourth in women in Iran. Eukaryotic release factor 3 (eRF3) is a GTPase associated with eRF1 in a complex that mediates translation termination in eukaryotes. Apart from its role in translation termination, the eukaryotic translation release factor 3 (eRF3) is involved in several critical cellular processes, such as cell cycle regulation and is essential for the G1 to S phase transition of the cell cycle (GSPT1), cytoskeleton organization, mRNA decay, recycle of ribosomes and apoptosis. Data from two previous studies on polymorphic GGC repeats located in exone 1 of the GSPT1/eRF3 gene suggest that this polymorphic site may play a role in cancer susceptibility.

The GGC expansion was amplified by PCR and alleles in variable size were selected by gelpolyacrylamid and sequenced directly. These sequenced alleles were used as allele specific markers.

In this research, we studies the GGC repeat polymorphism in GSPT1/eRF3 gene among 153 colorectal patients and 280 healthy controls in Isfahan region, Iran. Four different lengths of the GGC repeat in the range of 7,10,11,12 were observed. The most common genotype in controls and patients was homozygous with allele length of 10. Our primary data demonstrate that people carrying allele of 12 GGC specially 11/12 and 12/12 genotypes are at significantly higher risk of developing colorectal cancer.

A direct correlation has been found between the presence of the 12-Gly allele of this gene and development of colorectal cancer (OR = 2.66, P = 0.04). Our results also show a strong association between the allelic lengths of eRF3/GSPT1 polymorphism and family history of colorectal cancer (OR = 10.7, P = 0.0001).

HOXA1 is a transcription factor. In the human mammary gland, the expression of the HOXA1 gene is very low or absent during normal growth and differentiation, but it is expensed at high levels in breast cancer lesions. To date, there has been no study on the relationship between CAC repeat (polyhistidine) in the first exon of HOXA1 gene and the cancer risk. The purpose of this study was to investigate polymorphism of CAC in in the first exon of HOXA1 gene among patients with breast cancer and healthy individuals to clear the relationship of CAC polymorphism in HOXA1 Gene and the risk of breast cancer.

Peripheral blood samples were collected from 193 women with breast cancer and 200 healthy women. After DNA extraction from peripheral blood samples by salting-out method and amplification of desired sequence by polymerase chain reaction (PCR), the number of CAC repeat was determined by polyacrylamide gel electrophoresis and direct sequencing.

Five different length of CAC repeat in the range of 3-7 and 5 allele combinations (genotypes) were observed among patients and controls. The most frequent allele in both patients and controls was the 7-CAC repeat. No significant association was observed between this allele and breast cancer risk. Statistical analyses showed significant association between homozygote 7 and estrogen and progesterone receptors.

Our findings demonstrate that there is no significant association between CAC repeat in exon 1of HOXA1 gene and breast cancer risk. However, there is a significant association between homozygote 7 genotype and estrogen and progesterone receptors.

Genetic alternation in p53 gene is associated with tumorgenesis, especially in breast, colon and lung tumors. Somatic mutation of the p53 gene is the most common genetic alteration seen in human cancers. To our knowledge, before this research, there was no study on the relationship of TAAAA repeat in the first intron of p53 gene and cancer risk. The purpose of this study was to investigate the polymorphism of TAAAA in the first intron of p53 gene among patients with breast cancer and healthy individuals and its relation to risk of breast cancer.

Peripheral blood samples were collected from 200 women with breast cancer and 200 healthy women. After DNA extraction from peripheral blood samples via salting out method and amplification of desired sequence via polymerase chain reaction (PCR), the number of TAAAA repeats was determined using polyacrylamide gel electrophoresis and direct sequencing.

Five different length of TAAAA repeat in the range of 6-10 and 11 allele combinations (genotypes) were observed among patients and controls. The most frequent allele in both patients and controls was the 8-TAAAA repeat. Women who were homozygous for (TAAAA)7 or heterozygous for (TAAAA)6 were at higher risk of developing breast cancer. The allelic length of p53 polymorphisms had no significant effect on the metastasis, the expression of estrogen receptors, progesterone receptors and ErbB2 (HER2) and age of the onset.

Our study shows strong association between the TAAAA repeat polymorphism in p53 gene and risk of breast cancer.