Analysis of the methylation pattern of six gene promoters in sperm of men with abnormal protamination

It has recently been shown that alteration of the methylation pattern of imprinted genes is associated with different types of male infertility. The objective of our study was to investigate the methylation pattern of selected gene promoters in sperm of patients with abnormal protamine replacement. The promoters of OCT4, SOX2, NANOG, HOXC11, miR-17and CREMwere analyzed using bisulfite sequencing and the percentage of DNA methylation was compared between patients with an abnormal protamine l/protamine 2 （P1/ P2） ratio and normozoospermic controls. No significant quantitative differences were found between groups of patients with either an abnormally high or low P1/P2 ratio compared to normal controls. However, two individual samples from infertile subjects （2/20, 10%） showed an altered methylation pattern for the CREMgene promoter that was not found in control samples. These two samples had a significantly higher （P〈0.05） promoter methylation （5.58 and 4.23%, respectively） compared to the control group （0.46%）. In conclusion, in our pilot study, extreme methylations defects were not seen broadly in severely infertile men. However, two patients exhibited altered methylation of the CREMgene, which may be either causative or a result of abnormal protmaine replacement.

Myostatin gene promoter:structure,conservation and importance as a target for muscle modulation

Myostatin(MSTN) is one of the key factors regulating myogenesis. Because of its role as a negative regulator of muscle mass deposition, much interest has been given to its protein and, in recent years, several studies have analysed MSTN gene regulation. This review discusses the MSTN gene promoter, focusing on its structure in several animal species, both vertebrate and invertebrate. We report the important binding sites considering their degree of phylogenetic conservation and roles they play in the promoter activity. Finally, we discuss recent studies focusing on MSTN gene regulation via promoter manipulation and the potential applications they have both in medicine and agriculture.

The -250G>A polymorphism in the hepatic lipase gene promoter influences plasma lipid profile and lipoprotein ratio in patients with ischemic stroke

Objective:To evaluate the influence of–250G>A(rs2070895)polymorphism in hepatic lipase gene(LIPC)promoter on plasma lipid parameters of ischemic stroke patients.Methods:A total of 100 stroke patients and 100 control subjects matched for sex(59 men and 41 women)and age were selected.Hepatic lipase activity and lipid profiles were measured while lipoprotein ratios were calculated.Genotyping of the–250G>A promoter polymorphism of the LIPC was performed by the polymerase chain reaction and restriction fragment length polymorphism method combined with 2%gel electrophoresis and then confirmed by direct sequencing.The LIPC promoter gene sequencing data were compared with refseqNG011465.1 LIPC from GenBank.Results:The frequencies of GG,GA and AA genotypes of LIPC rs2070895 polymorphism were 39%,45%and 16%for the control,10%,37%and 53%for the stroke subjects(P<0.0001),respectively.The frequencies of G and A alleles were 61.5%and 38.5%for the control,and 28.5%and 71.5%for the stroke subjects(P<0.0001).Our study shows that the mutant allele of the LIPC promoter was associated with dyslipidemia,lower hepatic lipase activity,and this variation contributed to the increased defective plasma high-density lipoprotein-cholesterol(HDL-C),HDL2-C and HDL3-C concentration for both subjects.The control subjects had 6 single nucleotide polymorphism and 6 amino acid substitutions while the stroke subjects had 32 single nucleotide polymorphism and 20 amino acid substitutions.Conclusions:LIPC–250G>A polymorphism can influence plasma lipid profiles and lipoprotein ratios in patients with ischemic stroke.

Cloning and Sequence Analysis of SLC11A1 Gene Promoter of Three Cattle Breeds in Xinjiang

[Objective]Solute carrier family 11 member 1（SLC11A1）is a major natural resistance candidate gene,which contributes to defense mechanisms of a variety of intracellular bacteria.The SLC11A1 gene promoter sequence of Xinjiang Brown Cattle,Holstein and Simmental were cloned in the test,and promoter sequence difference was analyzed,in order to provide genetic marker-assisted selection for disease-resistant breeding of dairy cattle.[Method]The Genomic DNA was extracted from whole blood collected from three cattle breeds in Xinjiang,and the 5’ flanking region of SLC11A1 gene was amplified by PCR and sequenced.The sequence was analyzed by bioinformatics software CpGplot,RepeatMasker,TFSEARCH,WWW Signal Scan and dual luciferase assay system.[Result]The SLC11A1 gene promoter sequence of 1 463 bp was confirmed,which had promoter activity.No CpG islands were found on promoter sequence.There were four different sites in SLC11A1 gene promoter sequences between Angus from America and three cattle breeds in Xinjiang.Sequence analysis revealed 12 transcription factor binding sites including Sp1,NF1,RelA-p65,GKLF,and CPBP.In promoter region there was an enhancer region（-734- -740）and two short scattered repetitive elements BOV-tA2,MIR3,as well as repeated DNA element Charlie8.[Conclusion]The SLC11A1 gene promoter sequences of three breeds were obtained,which were different from that of Angus.The paper provided a theoretical basis for further studying the influence of SLC11A1 gene polymorphisms on resistance against intracellular bacteria infection.

Microcalorimetric Studies on Gene Promoter Function of Cloned DNA Fragements from Halobacterium halobium J7 Plasmid pHH205 in Escherichia coli TG1

Halobacterium halobium is a typical kind of extremely halophilic bacterium. Combined with the antibiotic resistance assay, the microcalorimetric method was used to study the promoter function of the cloned DNA fragments from Halobacterium halobium J7 plasmid pHH205 in Escherichia coli TG1. The promoter probe vector, plasmid pKK232-8, was used to form the recombinants. The DNA fragment, which is the promoter for the chloramphenicol acetyl transferase （CAT） gene in plasmid pKK232-8, is about 800 bp, and the chloramphenicol resistance level presented by IC50 is about 200 μg·mL^-1, which suggests a high promoter activity. The conclusions show that there probably exist double-function or trinary-function gene promoters in Halobacterium halobium, and Archaea may contain rich genetic resources.

Characterization of the Promoter of a Homolog of Maize MADS-Box Gene m18

Maize （Zea mays L.） is one of the world’s major food crops, and often suffers from tremendous yield loss caused by abiotic stresses. The MADS-box genes are known to play versatile roles in plants, controlling plant responses to multiple abiotic stresses. However, understanding of regulation of their expressions by the conventional loss-of-function approach is very dififcult. So far, regulation of MADS-box gene expression is little known. The best approach to retrieve expression regulation of this category of genes is to characterize expression of their promoters. In this study, the promoter of a homolog （GenBank accession no. EC864166） of maize MADS-box gene m18 was cloned by way of genome-walking PCR, named Pro66. Predicative analysis indicated that Pro66 contains more than one TATA box and multiple cis-acting environmental conditions-responsive elements （ECREs）. Pro66 could drive expression of theβ-glucuronidase （GUS）-encoding gene in maize, and heterologous expression of GUS in red pepper stressed by water deifcit, salt, copper, iron deifciency, heat, cold, and grown under short and long photoperiods, echoing predicative ECREs. Conclusively, maize MADS-box gene m18 likely plays versatile functions in maize response to multiple abiotic stresses due to the promoter with multiple cis-acting elements. The complex arrangement of multiple cis-acting elements in the promoter features meticulously regulated expression of m18. The results give informative clues for heterologous utilisation of the promoters in monocot and dicot species. The copy of the ECREs and heterologous expression of the promoter in dicot species are also discussed.

Cloning of Promoter of Chinese Bean GRP 1.8 Gene and Characterization of Its Function in Transgenic Tobacco Plants

In order to learn the expression pattern of GRP1 8(glycine rich protein) gene promoter in transgenic plants and to explore its potential application in plant genetic engineering for vascular specific expression of interested genes, GRP 1 8 promoter was amplified by PCR from Chinese bean genomic DNA. The intermediate vector was constructed by inserting vascular specific expression promoter of GRP 1 8 gene in vector pBI 101. The regenerated tobacco plants obtained were analyzed by PCR to select the putative transgenic plants. The histochemical localization of GUS( β D glucosidase) activity indicates that as for that of GRP 1 8 promoter we can confer the vascular specific expression of GUS gene.

Cloning, expression profiling and promoter functional analysis of bone morphogenetic protein 2 in the tongue sole(Cynoglossus semilaevis)

BMP2 plays crucial roles in vertebrate developmental process and acts as a bone inducer during osteogenesis. We present here the molecular cloning of bmp2 cDNA from the marine flatfish Cynoglossus semilaevis, and the analysis of bmp2 expression profiling and promoter function. The full length of bmp2 cDNA sequence is 2 048 bp,which encodes a protein of 422 amino acids. Tissue expression distribution of bmp2 was examined in 14 tissues of mature individuals by quantitative real time PCR（qRT-PCR）. The results revealed that bmp2 was expressed ubiquitously, and the highest expression level was detected in the spinal cord. Moreover, bmp2 expression levels were detected at 15 sampling time points of early developmental stages（egg, larva, juvenile and fingerling stages）.The highest expression level of bmp2 was observed at the gastrula stage, which was about ten times higher than those at the other three embryo stages. Whole-mount in situ hybridization showed that the bmp2 signal was strongly detected at the location of the crown-like larval fin, heart and liver, and slightly expressed in the notochord at one day post hatch（dph）; then the expression of bmp2 started to be concentrated in notochord at three dph. Subsequently, we characterized the 5′-flanking region of bmp2 by testing the promoter activity by Luciferase reporter assays. Positive regulatory region was detected at the location of –179 to ＋109. The predicted transcription factor binding sites（E-box binding factors, zinc finger transcription factor, etc.） in this region might participate in the transcriptional regulation of the bmp2 gene.

Genome-wide identification,phylogeny and expression analysis of the SBP-box gene family in maize(Zea mays)

The SQUAMOSA promoter binding protein （SBP）-box genes encode a kind of plant-specific transcription factors （TFs） and play important roles in the regulation of plant development. In this study, a genome-wide characterization of this family was conducted in maize （Zea mays）. Thirty-one SBP-box genes were identified to be distributed in nine chromosomes and 16 of them were complementary to the mature ZmmiR156 sequences. All the Z. mays SBP （ZmSBP） genes were classified into two clusters with eight subgroups according to the phylogenetic analysis of proteins, which were consistent with the pattern of exon-intron structures. The phylogenetic tree of the ZmSBP, Oryza sativa SBP-like （OsSPL） and Arabidopsis thaliana SBP-like （AtSPL） genes were constructed and all the SBP-box genes were divided into eight groups, which was the same as the classification of ZmSBP genes. The comparision of the expression profiles of all SBP-box genes in these three species indicated that most orthologous genes had similar expression patterns. The results from this study provided a basic understanding of the ZmSBP genes and might facilitate future researches for elucidating the SBP-box genes function in maize.

Transcriptional Regulation of Expression of the Maize Aldehyde Dehydrogenase 7 Gene (ZmALDH7B6) in Response to Abiotic Stresses

Aldehyde dehydrogenases（ALDHs） represent a large protein family, which includes several members that catalyze the oxidation of an aldehyde to its corresponding carboxylic acid in plants. Genes encoding members of the ALDH7 subfamily have been suggested to play important roles in various stress adaptations in plants. In this study, quantitative RT-PCR analysis revealed that a maize ALDH7 subfamily member（ZmALDH7B6） was constitutively expressed in various organs, including roots, leaves, immature ears, tassels, and developing seeds. The abundance of ZmALDH7B6 mRNA transcripts in maize roots was increased by ammonium, NaCl, and mannitol treatments. To further analyze tissue-specific and stress-induced expression patterns, the 1.5-kb 5′-flanking ZmALDH7B6 promoter region was fused to the β-glucuronidase（GUS） reporter gene and introduced into maize plants. In roots of independent transgenic lines, there was significant induction of GUS activity in response to ammonium supply, confirming ammonium-dependent expression of ZmALDH7B6 at the transcript level. Histochemical staining showed that GUS activity driven by the ZmALDH7B6 promoter was mainly localized in the vascular tissues of maize roots. These results suggested that ZmALDH7B6 is induced by multiple environmental stresses in maize roots, and may play a role in detoxifying aldehydes, particularly in vascular tissue.

The exploration of the correlation between the risk of obesity and the promoter methylation of PRDM16 gene

Objective To explore the association between the Cp G methylation level of positive regulatory domain containing 16(PRDM16)gene promoter and obesity or body mass index(BMI).Methods A total of 116 patients(91female adults and 25 male adults)with abdominal operation in a municipal hospital of Henan province were enrolled in this study and they were divided into

Identification of Conserved Regulatory Elements in Mammalian Promoter Regions:A Case Study Using the PCK1 Promoter

A systematic phylogenetic footprinting approach was performed to identify conserved transcription factor binding sites （TFBSs） in mammalian promoter regions using human, mouse and rat sequence alignments. We found that the score distributions of most binding site models did not follow the Gaussian distribution required by many statistical methods. Therefore, we performed an empirical test to establish the optimal threshold for each model. We gauged our computational predictions by comparing with previously known TFBSs in the PCK1 gene promoter of the cytosolic isoform of phosphoenolpyruvate carboxykinase, and achieved a sensitivity of 75% and a specificity of approximately 32% Almost all known sites overlapped with predicted sites, and several new putative TFBSs were also identified. We validated a predicted SP1 binding site in the control of PCK1 transcription using gel shift and reporter assays. Finally, we applied our computational approach to the prediction of putative TFBSs within the promoter regions of all available RefSeq genes. Our full set of TFBS predictions is freely available at http：//bfgl.anri.barc.usda.gov/tfbsConsSites.

Genomic analyses of metal resistance genes in three plant growth promoting bacteria of legume plants in Northwest mine tailings, China

To better understand the diversity of metal resistance genetic determinant from microbes that survived at metal tailings in northwest of China, a highly elevated level of heavy metal containing region, genomic analyses was conducted using genome sequence of three native metal-resistant plant growth promoting bacteria（PGPB）. It shows that： Mesorhizobium amorphae CCNWGS0123 contains metal transporters from P-type ATPase, CDF（Cation Diffusion Facilitator）, Hup E/Ure J and CHR（chromate ion transporter） family involved in copper, zinc, nickel as well as chromate resistance and homeostasis. Meanwhile, the putative Cop A/Cue O system is expected to mediate copper resistance in Sinorhizobium meliloti CCNWSX0020 while Znt A transporter, assisted with putative Czc D, determines zinc tolerance in Agrobacterium tumefaciens CCNWGS0286. The greenhouse experiment provides the consistent evidence of the plant growth promoting effects of these microbes on their hosts by nitrogen fixation and/or indoleacetic acid（IAA） secretion,indicating a potential in-site phytoremediation usage in the mining tailing regions of China.