Analysis of Growth Characteristics and Differentially Expressed Homologous Genes in Rhodobacter sphaeroides under Normal and Simulated Microgravity Conditions

The term “microgravity” is used to describe the “weightlessness” or “zero-g” circumstances that can only be found in space beyond earth’s atmosphere. Rhodobacter sphaeroides is a gram-negative purple phototroph, used as a model organism for this study due to its genomic complexity and metabolic versatility. Its genome has been completely sequenced, and profiles of the differential gene expression under aerobic, semi-aerobic, and photosynthetic conditions were examined. In this study, we hypothesized that R. sphaeroides will show altered growth characteristics, morphological properties, and gene expression patterns when grown under simulated microgravity. To test that, we measured the optical density and colony-forming units of cell cultures grown under both microgravity and normal gravity conditions. Differences in the cell morphology were observed using scanning electron microscopy (SEM) images by measuring the length and the surface area of the cells under both conditions. Furthermore, we also identified homologous genes of R. spheroides using the differential gene expression study of Acidovorax under microgravity in our laboratory. Growth kinetics results showed that R. sphaeroides cells grown under microgravity experience a shorter log phase and early stationary phase compared to the cells growing under normal gravity conditions. The length and surface area of the cells under microgravity were significantly higher confirming that bacterial cells experience altered morphological features when grown under microgravity conditions. Differentially expressed homologous gene analysis indicated that genes coding for several COG and GO functions, such as metabolism, signal-transduction, transcription, translation, chemotaxis, and cell motility are differentially expressed to adapt and survive microgravity.

Identification and Comparative Analysis of DGAT1 Genes among Cotton and Other Model Plants

Diacylglycerol acyltransferase （DGAT） is the key enzyme that catalyzes the triacylglycerol biosynthesis. The comparative analysis was performed on the DGAT1 genes of cotton and other model plants. Sequence analysis showed that most of the DGAT1 genes,with high variation of intron length and high conservation of intron phrase,from the cotton and other model plants had 16 exons. Additionally, 7 conserved motifs were present in these DGAT1 proteins. The core motifs were overlapped with the functional domain of DGAT1 protein. Phylogenetic analysis demonstrated that gene tree was highly consistent to species tree,suggesting that the evolutionary history of species was revealed by gene tree. There was single copy of DGAT1 gene in cotton,but at least two duplicated DGAT1 genes were iden- tified in rice,maize,poplar and moss genomes. The selective pressure analysis showed that the PtDGATla/PtDGATlb was under positive selection,but other four pairs of homologous genes were under negative selection. 17 positively selected sites were identified at subgroup level （P〉0.05）,suggesting these subgroups under relaxed functional constraint. The findings provide a basis for further studying func- tion and evolution of DGAT1 genes in cotton and other model plants.

Cloning and Analysis of a Disease Resistance Gene Homolog from Soybean

Conserved domains e.g. nucleotide binding site (NBS) were found in several cloned plant disease resistance genes. Based on the NBS domain, resistance gene analogs (RGAs) have been isolated previously and were used as probes to screen a soybean (Glycine max L. Merr.) cDNA library. A full-length cDNA, KR3, was obtained by screening the library and rapid amplification of cDNA ends (RACE) method. Sequence analysis revealed that the cDNA is 2 353 bp in length and the open reading frame (ORF) codes for a polypeptide of 636 amino acids with a Toll-Interleukin-1 receptor (TIR) and a NBS domain. Sequence alignment showed that it was similar to N gene of tobacco. The phylogenetic tree analysis of R proteins with NBS from higher plants was performed. The KR3 gene has low copies in soybean genome and its expression was induced by exogenous salicylic acid (SA).

Prognostic role of sensitive-to-apoptosis gene expression in rectal cancer

AIM:To investigate the association between prognosis of rectal cancer treated with chemoradiotherapy(CRT) and expression of sensitive-to-apoptosis(SAG),B-cell lymphoma-extra large(Bcl-X L) and Bcl-2 homologous antagonist/killer(Bak).METHODS:Real-time quantitative polymerase chain reaction was used to determine the expression of proteins of interest,namely SAG,Bcl-X L,Bak and β-actin,in rectal carcinoma patients who had a follow-up period of 3 years after CRT.Biopsy specimens were excised from the rectal tumor preceding CRT.RESULTS:SAG,Bcl-X L and Bak proteins showed significant correlations with each other.In multivariate analysis,patients with high vs low SAG expression showed a statistically significant difference in 2-year survival rates:56% vs 73%,respectively(P = 0.056).On the other hand,there were no significant correlations between the expression levels of all three genes and metastatic rates or tumor responses to CRT.Mean overall survival in the patients with elevated SAG expression was 27.1 mo ± 3.9 mo [95% confidence interval(CI):19.3-34.9],and in patients with reduced expression,it was 32.1 mo ± 2.5 mo(95% CI:27.3-36.9).The corresponding values for Bcl-X L were 28.0 mo ± 4.1 mo(95% CI:19.9-36.1) and 31.7 mo ± 2.9 mo(95% CI:26.0-37.5),and those for Bak were 29.8 mo ± 3.7 mo(95% CI:22.5-37.2) and 30.6 mo ± 2.4 mo(95% CI:25.5-35.0),respectively.CONCLUSION:Two-year survival rates significantly correlated with low SAG expression,and SAG may be a candidate gene for good prognosis,independent of therapeutic response of different individuals.

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.

Point mutations of homologs as an adaptive solution to the gene loss

Gene loss is common and influences genome evolution trajectories.Multiple adaptive strategies to compensate for gene loss have been observed,including copy number gain of paralogous genes and mutations in genes of the same pathway.By using the Ubl-specific protease 2(ULP2)eviction model,we identify compensatory mutations in the homologous gene ULP1 by laboratory evolution and find that these mutations are capable of rescuing defects caused by the loss of ULP2.Furthermore,bioinformatics analysis of genomes of yeast gene knockout library and natural yeast isolate datasets suggests that point mutations of a homologous gene might be an additional mechanism to compensate for gene loss.

Detection of hMSH2 and hMLH1 mutations in Chinese hereditary non-polyposis colorectal cancer kindreds

AIM： To establish and validate the mutation testing for identification and characterization of hereditary non-polyposis colorectal cancer （HNPCC） in suspected Chinese patients. METHODS： Five independent Chinese kindreds with HNPCC fulfilling the classical Amsterdam criteria were collected. Genomic DNA was extracted after informed consent was obtained. The coding region of hMSH2 and hMLH1 genes was detected by polymerase chain reaction （PCR） and denaturing high-performance liquid chromatography （DHPLC）. Mutations identified in the proband by DHPLC were directly sequenced using a 377 DNA sequencer, analyzed with a basic local alignment tool （BLAST）, and tested in the corresponding family members by direct DNA sequencing. RESULTS： Mutations were identified in two Chinese HNPCC kindreds. One was the missense mutation of hMSH2 c.1808A→G resulting in Asp 603 Gly identified in the proband of the fifth HNPCC （HNPCCS） kindred. In the HNP5 kindred, three family members were found to have this mutation and two of them had colorectal cancer. The other mutation of hMLH1 c.1882A→G was identified in the HNP2 kindred＇s proband, which might be the nonsense mutation analyzed by BLAST. CONCLUSION： Pedigree investigation and mutation testing of hMSH2 and hMLH1 are the practical methods to identify high-risk HNPCC patients in China.

Effects of controlled superovulation on the expression of HOXA10,integrin αvβ3 and LIF in mice during peri-implantation

Objective:To observe the expression of endometrial homologous box gene A10(HOXA10),Integrinαvβ3 and leukemia suppressor factor(LIF)in peri-implantation mice,and to investigate the effect of controlled superovulation(COH)on endometrial receptivity in mice.Methods:After COH model preparation and blastocyst transplantation,kunming pure-bred mature mice were randomly divided into natural blastocyst+natural pseudocyst group,COH blastocyst+natural pseudocyst group,natural blastocyst+COH pseudocyst group,and COH blastocyst+COH pseudocyst group.The pregnancy rate and average number of beds in each group were observed on day 6 of conception.Western blot and RT-PCR were used to detect the expression of HOXA10,Integrinααvβ3,LIF protein and mRNA in the endometria of mice on day 5 of conception.Results:The pregnancy rate and average number of beds in the COH group were lower than those in the natural receptor group.The expression of HOXA10,Integrinααvβ3 and LIF in endometrium was significantly lower than that of the natural receptor mice(P<0.01).Conclusion:Superovulation drugs down-regulate the expression of HOXA10,Integrinαvβ3 and LIF in endometrium during the implantation window,reduce endometrial receptivity,and lead to low clinical pregnancy rate.

Salinity tolerance in barley during germination——homologs and potential genes

Salinity affects more than 6%of the world’s total land area,causing massive losses in crop yield.Salinity inhibits plant growth and development through osmotic and ionic stresses;however,some plants exhibit adaptations through osmotic regulation,exclusion,and translocation of accumulated Na+or Cl-.Currently,there are no practical,economically viable methods for managing salinity,so the best practice is to grow crops with improved tolerance.Germination is the stage in a plant’s life cycle most adversely affected by salinity.Barley,the fourth most important cereal crop in the world,has outstanding salinity tolerance,relative to other cereal crops.Here,we review the genetics of salinity tolerance in barley during germination by summarizing reported quantitative trait loci(QTLs)and functional genes.The homologs of candidate genes for salinity tolerance in Arabidopsis,soybean,maize,wheat,and rice have been blasted and mapped on the barley reference genome.The genetic diversity of three reported functional gene families for salt tolerance during barley germination,namely dehydration-responsive element-binding(DREB)protein,somatic embryogenesis receptor-like kinase and aquaporin genes,is discussed.While all three gene families show great diversity in most plant species,the DREB gene family is more diverse in barley than in wheat and rice.Further to this review,a convenient method for screening for salinity tolerance at germination is needed,and the mechanisms of action of the genes involved in salt tolerance need to be identified,validated,and transferred to commercial cultivars for field production in saline soil.

ZFN,TALEN and CRISPR-Cas9 mediated homology directed gene insertion in Arabidopsis:A disconnect between somatic and germinal cells

Breakthroughs in the generation of programmable sequence-specific nucleases (SSNs), such as zinc finger nucleases (ZFNs),TAL effector nucleases (TALENs) and the RNA-directed nuclease CRISPR-associated protein 9 (Cas9), have greatly increased the ease of plant genome engineering (Voytas, 2013; Malzahn et al.,2017). Programmable SSNs introduce a DNA double-strand break

A Novel Mutation of Notch homolog protein 2 gene in a Chinese Family with Hajdu-Cheney Syndrome

Hajdu-Cheney syndrome （HCS） is a rare disorder which is characterized by developmental delay, craniofacial anomalies, congenital heart defects, hearing deficit, polycystic kidneys, and bone abnormalities, including progressive osteoporosis, acroosteolysis, wormian bones, and abnormal bonefractures.

TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice

Over the last decades,much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology.The breakthrough has been made in recent years with the advent of sequence-specific endonucleases,especially zinc finger nucleases（ZFNs）,transcription activator-like effector nucleases（TALENs） and clustered regularly interspaced short palindromic repeats（CRISPRs） guided nucleases（e.g.,Cas9）.In higher eukaryotic organisms,site-directed mutagenesis usually can be achieved through non-homologous end-joining（NHEJ） repair to the DNA double-strand breaks（DSBs） caused by the exogenously applied nucleases.However,site-specific gene replacement or genuine genome editing through homologous recombination（HR） repair to DSBs remains a challenge.As a proof of concept gene replacement through TALEN-based HR in rice（Oryza sativa）,we successfully produced double point mutations in rice acetolactate synthase gene（OsALS） and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations.After ballistic delivery into rice calli of TALEN construct and donor DNA,nine HR events with different genotypes of OsALS were obtained in T_0 generation at the efficiency of 1.4%—6.3%from three experiments.The HRmediated gene edits were heritable to the progeny of T_1 generation.The edited T_1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance.The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.

Molecular characteristics of the omp A gene of serotype B Chlamydia trachomatis in Qinghai Tibetan primary school students

To study the molecular characteristics of Chlamydia trachomatis, the major outer membrane protein gene(omp A) of C. trachomatis from primary school students with trachoma residing in the Qinghai Tibetan area was sequenced and compared with the same serotype in Gen Bank. In Jianshetang Primary School and Galeng Central Primary School in the Galeng Tibetan Township of Qinghai Haidong Sala Autonomous County, scraped samples were collected from the upper tarsal conjunctiva and lower conjunctival sac of both eyes of 45 students with trachoma, stored at 4°C, and transported to Beijing Tongren Hospital by air within 24 h. The samples were screened for C. trachomatis by real-time PCR. The omp A gene from the C. trachomatis-positive samples was amplified by nested PCR. The serotype was confirmed by National Center for Biotechnology Information(NCBI) BLAST search and homology analysis. The entire omp A gene sequence was compared with the corresponding gene sequences of serotype B strains available in Gen Bank. Of the 45 students aged 6–13 years with trachoma, 26 C. trachomatis-positive students were identified by the initial real-time PCR screening(average age,(9.09±1.63) years; sex ratio, 1.0), accounting for 57.78%(26/45). The cycle threshold values for real-time PCR were 16.79–37.77. Half(13/26) of C. trachomatis-positive students had a bacterial copy number of >105. The compliance rate of the omp A gene sequences with the C. trachomatis serotype B strains in Gen Bank was up to 99%. Two novel genetic mutations were found when the omp A gene was compared with those of the 11 serotype B strains in Gen Bank. The two non-synonymous mutations were located at(i) position 271 in the second constant domain, an adenine(A) to guanine(G) substitution(ACT?GCT), changing the amino acid at position 91 from threonine to alanine(Thr?Ala) in all 26 strains; and(ii) position 887 in the fourth variable domain, a cytosine(C) to thymine(T) substitution(GCA?GTA), changing the amino acid at residue 296 from alanine to valine(Ala?Val) in four of the 26 strains. Six mutations were identified relative to ATCC VR-573. The strains could be divided into two gene clusters according to the mutation at nucleotide position 887: CQZ-1(China Qinghai Tibetan-1) and CQZ-2(China Qinghai Tibetan-2). We thus detected two novel serotype B mutant strains of C. trachomatis among study subjects with trachoma.