Abnormal Change of p53 Gene in Gastric and PrecancerousLesions and APC Gene Deletion in Gastric Carcinoma and Near Tissues

p53 gene mutation (exon4, 5, 6, 7, 8 and intron6) in gastric cancer and precancerous lesions and p53 gene (exon4 and ontron6), APC gene deletion in gastric carcinomas were studied by PCR/SSCP and PCR/RFLP- Results showed mutation rate of p53 in metaplasia, dysplasia and gastric carcinoma was 37. 5 % (3/8), 42. 11 % (8/19), 53. 33 (16/30) respectively- There was significant dif-ference among groups of metaplasia, dysplasia, cancer and normal controls. Noexon8 mutation was found in metaplasia and dysplasia, but 4 cases were found to have exon8 mutation in cancer group. It is suggested that exon8 mutation occurs at the late stage of gastric cancer, but exon 5, 6, 7 mutation occur in the course ofprecancerous lesion to cancer. Loss of heterozygosity (LOH) of exon4, intron6,APC was 47,37 % (9/19), 8. 73% (2/23), 16. 67 % (3/18) respectively. LOH of exon4 had something to do with poor differentiation, lymph node metastasis,depth of invasion- LOH of exon4 may be one of prognostic marker of gastric cancer. We are led to conclude that p53 gene mutation is an early event and perhaps work together with ras oncogene in gastric carcinogenesis

DAZ1/DAZ2 cluster deletion mediated by gr / gr recombination per se may not be sufficient for spermatogenesis impairment： a study of Chinese normozoospermic men

Aim： To explore the possible effect of the deleted in azoospermia （DAZ） copy cluster deletion on spermatogenesis in the Chinese population, the deletion of the azoospermia factor c （AZFc） region was analyzed in 346 normozoospermic men. Methods： Three DAZ single nucleotide variant loci and seven AZFc-specific sequence-tagged sites were examined with polymerase chain reaction （PCR）-restriction fragment length polymorphism and routine PCR. Results： Five （1.4%） of the normozoospermic men were found to have deletion of grlgr-DAZ1/DAZ2. None of the men were found to have b2/b4--entire DAZ deletion. Conclusion： The presence of grlgr-DAZ1/DAZ2 deletion in five men with normozoospermia suggests that this deletion per se may not be sufficient for spermatogenic impairment in Chinese men. （Asian J Androl 2006 Mar; 8： 183-187）

Development of a pyrF-based counterselectable system for targeted gene deletion in Streptomyces rimosus

Streptomyces produces many valuable and important biomolecules with clinical and pharmaceutical applications.The development of simple and highly efficient gene editing tools for genetic modification of Streptomyces is highly desirable.In this study,we developed a screening system for targeted gene knockout using a uracil auxotrophic host(ΔpyrF)resistant to the highly toxic uracil analog of 5-fluoroorotic acid(5-FOA)converted by PyrF,and a non-replicative vector pKC1132-pyrF carrying the complemented pyrF gene coding for orotidine-5'-phosphate decarboxylase.The pyrF gene acts as a positive selection and counterselection marker for recombinants during genetic modifications.Single-crossover homologous integration mutants were selected on minimal medium without uracil by reintroducing pyrF along with pKC1132-pyrF into the genome of the mutantΔpyrF at the targeted locus.Double-crossover recombinants were generated,from which the pyrF gene,plasmid backbone,and targeted gene were excised through homologous recombination exchange.These recombinants were rapidly screened by the counterselection agent,5-FOA.We demonstrated the feasibility and advantage of using this pyrF-based screening system through deleting the otcR gene,which encodes the cluster-situated regulator that directly activates oxytetracycline biosynthesis in Streptomyces rimosus M4018.This system provides a new genetic tool for investigating the genetic characteristics of Streptomyces species.

FREQUENT DELETION OF MTS1/p16 GENE AND CORRELATION WITH CLINICOPATHOLOGICAL PARAMETERS IN ENDOMETRIAL CARCINOMA

Objective: To investigate the possible relationship between deletion of MTS/p16 gene and progression of endometrial carcinoma Methods: Forty six primary endometrial carcinoma, 7 tumor adjacent endometrial tissue, 10 normal endometrial tissue specimen and 5 xenografts from patients with endometrial carcinoma were examined for homozygous deletion of MTS/p16 gene by polymerase chain reaction based analysis Results: Of 46 endometrial cancer specimens, 9 showed homozygous deletion, no deletion was detected in the tumor adjacent and normal endometial tissues Nor was it detected in well differentiated endometrial carcinoma and all xenografts Conclusions: Deletion of MTS1/p16 gene might contribute to the progression of endometrial carcinoma and could be served as indicator for predicting prognosis

A VNTR ELEMENT ASSOCIATED WITH STEROID SULFATAES GENE DELETIONS STIMULATES RECOMBINATIONIN CULTURED CELLS

Steroid sulfatase deficiency is a common genetic disorder, with a prevalence of approximately one in every 3500 males world wide.About 90% of these patients have complete gene deletions, which appear to result from recombination between members of a low-copy repeat family (CR1-232 is the prototype) that flank the gene. RUI and RU2 are two VNTR elements found within each of these family members.The RU1 consists of 30bp repeating units and its length shows minimal variation among individuals. The RU2 element consists of repeating sequences which are highly asymmetric, with about 90% purines and no C's on one strand, and range from 0. 6kb to over 23kb among different individuals. We conducted a study to determine if the RU1 and RU2 elements can promote recombination in an in vivo test system.We inserted these elements adjacent to the neo gene in each of two pSV2neo derivatives. One of which has a deletion in the 5' portion of neo gene and the other having a deletion in the 3'portion. These plasmids were combined and used to transfect EJ human bladder tumor cells. Survival of cells in G418 indicates restoration of a functional neo gene by recombination between two deletion constructs. Thus counting G418 resistant colonies gives a quantitative measure of the enhancement of recombination by the inserted VNTR elements.The results showed no effect on recombination by the inserted RU1 element(compared to the insertion of a nonspecific sequence), while the RU2 element stimulated.recombination by 3. 5-fold (P< 0.01). A separate set of constructs placed RU1 or RU2 within the nitron of an exon trapping vector. Following transfection of cells, recombination events were monitored by a quantitative PCR assay that detected the approximation of primer banding sites (as a result of recombination).These studies showed that, as in the first set of experiments, the highly variable RU2 element is capable of stimulating somatic recombination in tnammalian cells.

DELETION AND INACTIVATION OF RETINOBLASTOMA SUSCEPTIBILITY GENE IN PRIMARY RETINOBLASTOMA

The status and expression of Rb gene was detected and analyzed in 19 surgical retinoblastoma specimens using Rb cDNA 3. 8 kb and 0. 9 kb fragment as probe and antibodies specific for synthetic Rb peptide or expressive product of Rb gene expression plasmld. DNA from those tumors had the hemlzygous deletion in 3 cases, the homozygous internal deletion In 2 cases and alterated restriction fragment involving In one copy of Rb gene In 1 case. The quantity of Rb protein demonstrated either absence of reduction in all the 16 cases examined In comparison with that in normal adult retina. It suggested that there were structural or/ and functional defects of Rb gene In retinoblastoma cells and provided evidence to support Knudson' s two hit hypothesis.

Expression of Porcine Interleukin-2 and Porcine Interleukin-6 and Their Adjuvant Effects on Gene Deleted Vaccine of Pseudorabies Virus(TK^-/gG^-/LacZ^+)

Porcine interleukin-2 and porcine interleukin-6 cDNA sequences were cloned into the expressing vectors pET-28a and pGEX-KG respectively. They were expressed in E. coli BL21(DE3)with high-level production. The gene deleted vaccine of pseudorabies virus Ea strain(TK-/gG-/LacZ+)was mixed with the two different purified recombinant proteins each, or both, with the doses of 2, 5 or 10 μg ml-1. Ten groups of pseudorabies negative antibody swines were immuned twice with tested vaccines with different doses, or control vaccine, respectively. The antibody liters of the test groups were detected by neutralization test, and the daily weight gains of swines were calculated and analyzed statistically. In the study, all the neutralizing antibody ti-ters in test groups were higher than the control group, and the recombinant proteins appeared a dose dependent adjuvant effect. The tested vaccines with 2 μg ml-1 pIL-2 and with 10 μg ml-1 pIL-2/pIL-6 got significant and extremely significant differences, compared with the vaccines without pILs. The difference of the daily weight gain indicated the potential positive influence of pIL-2 and pIL-6 on immune protection.

Cre-recombinase systems for induction of neuronspecific knockout models:a guide for biomedical researchers

Gene deletion has been a valuable tool for unraveling the mysteries of molecular biology.Early approaches included gene trapping and gene targetting to disrupt or delete a gene randomly or at a specific location,respectively.Using these technologies in mouse embryos led to the generation of mouse knocko ut models and many scientific discoveries.The efficacy and specificity of these approaches have significantly increased with the advent of new technology such as cluste red regula rly inters paced short palindromic repeats for targetted gene deletion.However,several limitations including unwanted off-target gene deletion have hindered their widespread use in the field.Crerecombinase technology has provided additional capacity for cell-specific gene deletion.In this review,we provide a summary of currently available literature on the application of this system for targetted deletion of neuronal genes.This article has been constructed to provide some background info rmation for the new trainees on the mechanism and to provide necessary information for the design,and application of the Cre-recombinase system thro ugh reviewing the most f requent promoters that are currently available for genetic manipulation of neuro ns.We additionally will provide a summary of the latest technological developments that can be used for targeting neurons.This may also serve as a general guide for the selection of appropriate models for biomedical research.

Phosphatase and tensin homology deleted in chromosome 10,hypoxia-inducible factor-1 alpha gene expression in colorectal adenoma and adenocarcinoma and their relation to vascular endothelial growth factor protein expression

To examine phosphatase and tensin homology deleted in chromosome 10 (PTEN),hypoxia-inducible factor-1 alpha (HIF-1 alpha) gene expressions and their relation to vascular endothelial growth factor(VEGF) protein expression in the patients with human colorectal adenomas and adenocarcinomas.Methods The expression of PTEN,HIF-1 alpha gene was detected by using in situ hybridization,and the VEGF expression levels by immunohistochemistry in colorectal adenomas and primary colorectal adenocarcinoma.Results Strong expression of HIF-1 alpha was detectable in the majority of colorectal dadenocarcinoma,particularly surrounding areas of necrosis in adenocarcinoma.PTEN,HIF-1 alpha mRNA and VEGF protein were positive in 51.6%,67.7% and 59.7% respectively in 62 cases of adenocarcinomas,and 77.8%,44.4% and 33.3% respectively in 18 cases of adenomas.The positive rate of VEGF was higher in the patients with colorectal adenocarcinomas than that in those with adenomas,whereas that of PTEN mRNA was contrary.HIF-1 mRNA expression was correlated significantly with lymph node metastasis,liver metastasis,Duke’s stage and recurrence.During colorectal tumor progression,the expression of HIF-1 alpha mRNA was positively correlated with the VEGF protein expression (χ2= 4.751 ,P<0.05),but negatively with the PTEN mRNA expression(χ2=21.84,P<0.01).Conclusion The absence or low expression of PTEN and the increased levels of HIF-1α and VEGF may paly an important role in carcinogenesis and progression of colorectal carcinoma.These results suggest that VEGF upregulated by HIF-1 alpha gene may be involved in angiogenesis of colorectal adenocarcinoma.4 refs,1 tab.

High risk genetic factor in Chinese patients with idiopathic male infertility: deletion of DAZ gene copy on Y chromosome

Idiopathic azoospermia or oligozoospermia affects approximately 2% -4% of allmarried males. Recently studies have confirmed that the deletion of DAZ in AZFc region of Ychromosome may be one of the important genetic aetiologies of Caucasian male infertility. Todetermine the relationship between DAZ gene deletion and idiopathic male infertility in Chinesepopulation, we analysed the DAZ gene copy number of AZFc region in patients with idiopathicazoospermia or oligozoospermia, as well as fertile Chinese men.

Prevalence of Plasmodium falciparum isolates lacking the histidine rich protein 2 gene among symptomatic malaria patients in Kwilu Provinee of the Democratic Republic of Congo

Background:Malaria rapid diagnostic tests have become a primary and critical tool for malaria diagnosis in malariaendemic countries where Plasmodium falciparum Histidine Rich Protein 2-based rapid diagnostic tests(PflHRP2-based RDTs)are widely used.However,in the last decade,the accuracy of PflHRP2-based RDTs has been challenged by the emerge nee of P.falciparum strains harbouring deletions of the P.falciparum histidine rich protein 2(pflnrp2)gene,resulting in false-negative results.In the Democratic Republic of Congo(D.R.Congo),little is known about the prevalence of the pfhrp2 gene deletion among P.falciparum isolates infecting symptomatic patients,especially in low to moderate transmission areas where pfhrp2 deletion parasites are assumed to emerge and spread.Here we determine the local prevalence and factors associated with pfhrp2 gene deletions among symptomatic malaria patients in the Kwilu Province of the D.R.Congo.

Is the human dystrophin gene's intron structure related to its intron instability?

Objective To study the human dystrophin gene molecular deletion mechanism, we analyzed breakpoint regions within junction fragments of deletion-type patients and investigated whether the dystrophin gene's intron structure might be related to intron instability.Methods Junction fragments corresponding to exon 46 and 51 deletions were cloned. The breakpoint regions were sequenced, and the features of introns with available Genebank sequences were analyzed.Results An analysis of junction fragment sequences corresponding to exon 46 and 51 deletions showed that all 5' and 3' breakpoints are located within repeat sequences. No small insertions, small deletions, or point mutations are located near the breakpoint junctions. By analyzing the secondary structure of the junction fragments, we demonstrated that all junction fragment breakpoints are located in non-matching regions of single-stranded hairpin loops. A high concentration of repetitive elements is found to be a key feature of many dystrophin introns. In total, 34. 8% of the overall dystrophin intron sequences is composed of repeat sequences.Conclusion Repeat elements in many dystrophin gene introns are the key to their structural bases and reflect intron instability. As a result of the primary DNA sequences, single-stranded hairpin loops form, increasing the instability of the gene, and forming the base for breaks in the DNA. The formation of the single-stranded hairpins can result in reattachment of two different breakpoints, producing a deletion.

Analysis of Chinese women with primary ovarian insufficiency by high resolution array-comparative genomic hybridization

Background Primary ovarian insufficiency （POI） is defined as a primary ovarian defect characterized by absent menarche （primary amenorrhea） or premature depletion of ovarian follicles before the age of 40 years. The etiology of primary ovarian insufficiency in human female patients is still unclear. The purpose of this study is to investigate the potential genetic causes in primary amenorrhea patients by high resolution array based comparative genomic hybridization （array-CGH） analysis. Methods Following the standard karyotyping analysis, genomic DNA from whole blood of 15 primary amenorrhea patients and 15 normal control women was hybridized with Affymetrix cytogenetic 2.7M arrays following the standard protocol. Copy number variations identified by array-CGH were confirmed by real time polymerase chain reaction.

Analysis of Distribution of Thalassemic Genotype and Clinical Phenotype

Objective To determine the incidence of Thalassemia, the distribution of Thalassemic genotype and clinical phenotype. Methods Reverse dot blot （RDB） hybridization was used to detect the common three a-globin gene deletions（--SEA, -a3.7 and-a4.2） by Gap-PCR over all 8 118 cases and to detect 17 fl-globin gene 17 mutations （CD41-42, IVS- ][-654, CD17, -28, fiE, CD71- 72, CD27-28, -29, CD43, CD14-15, IVS-I-1, IVS-I-5, CAP, -31, Int, -32, -30） among 7 934 eases. Patients were grouped according to clinical phenotype such as anemia symptom, screening test of thalassemia and family carrier history. Results Among 8 118 cases, there were 2 519 cases with a-globin gene deletions over 9 kinds genotypes, and the incidence ofa-thal was 31.03%. The genotypes of--SEA/aa, -a3.7/aa, -a4.2/aa, -a3.7/--SEA and -a4.2/-sEA were common and constituent ratios were 77.05%, 11.95%, 4.01%,3.65% and 2.10%, respectively, and altogether was 98. 76%. Over all 7 934 cases, there were 1 691 cases with fl-globin gene mutation over 13 kinds genotypes, and the incidence of fl-thal was 21.31%, The mutation of CD41-42, IVS- ff-654, CD17 and -28 were common, and constituent ratios were 34.24%, 29.80%, 16.03% and 10. 70%, respectively, and altogether was 90. 77%. Number of patients with screening test positive was the largest, and the incidence of the group with three indications was the highest. The abnormal percentage in group both with anemia symptom and screening test positive was the highest. Conclusion There was a high thalassemia carrier rate among patients with clinical indications in our study. The main indication for diagnosis of thalassemia was both with anemia symptoms and screening test positive. The characteristics of thalamessia genotype in our study were consistent with that in southern China. It was important for population, especially reproductive population of high frequency area region to screen- ing and diagnosing thalassemia.