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Copy Number Variation (CNV) is the major manner for the variation of genome structure, which is associated with numerous important traits. The heat shock factor 1 (HSF1) gene is a stress response transcriptional regulator. It participates in the heat shock response, simultaneously participated in the development of tissue. The objective of this research was to explore the influence of CNV of the HSF1 gene on the growth traits of the Ashidan yak. In this study, the growth traits (withers height, body weight, chest girth, and body length) of 274 Ashidan yaks were divided into four stages (6, 12, 18, and 30 months old). Moreover, quantitative polymerase chain reaction (qPCR) was exploited for determining the HSF1 gene relative expression level, and SPSS software was utilized for the statistical analysis. The outcomes indicated that HSF1-CNV was significantly associated with body length (p 0.05) and was extremely significant associated with withers height (p 0.01) of 18-month-old Ashidan yaks. Besides, the HSF1 relative expression in heart and muscle was higher than that existed in other tissues (p 0.01). The outcomes suggested that the CNV of HSF1 gene would affect the growth and development of the Ashidan yak, which is conducive to the early breeding of yak.

Previous, studies have shown that the dynein transporter compound has a role in diseases such as intellectual disability and cerebral malformations. However, the study of CNV in DYNC1I2 gene has not been reported. Q-PCR and data association analysis were used for DYNC1I2 gene copy in this study. In this study, blood samples were collected from five breeds of Chinese cattle (Qingchuan cattle, Xianan cattle, Yunling cattle, Pinan cattle and Guyuan cattle) for DYNC1I2 gene CNV type detection. SPSS 20.0 software and method of ANOVA were used to analyzed the association between types of CNV and growth traits. Results reveal that the distribution of different copy number types in different cattle breeds is different. Association analysis indicate that CNV of DYNC1I2 gene showed a positive effect in cattle growth: in XN cattle, individuals with deletion types showed better performance on height at hip cross (P

Copy number variations (CNVs) recently have been recognized as another important genetic variability followed single nucleotide polymorphisms (SNPs). The guanylate binding protein 2 (GBP2) gene plays an important role in cell proliferation. This study was performed to determine the presence of GBP2 CNV (relative to Angus cattle) in 466 individuals representing six main cattle breeds from China, identify its relationship with growth, and explore the biological effects of gene expression. There were two CNV regions in the GBP2 gene, for three types, CNV1 loss type (relative to Angus cattle) was more frequent in XN than other breeds, and CNV2 loss type (relative to Angus cattle) was more frequent in XN and CDM than other breeds. Though the GBP2 gene copy number presented no correlation with the transcriptional expression of JX (P .05), but the transcriptional expression in heart is higher than other tissues, and the copy number in muscles and fat of JX is higher than others breeds. Statistical analysis revealed that the GBP2 gene CNV1 and CNV2 were significantly associated with growth traits (P .05). In conclusion, this research established the correlations between CNVs of GBP2 gene and growth traits in different cattle breeds, and our results suggested that the CNVs in GBP2 gene may be considered markers for the molecular breeding of Chinese beef cattle.

The number of active ribosomal genes (AcRG) was evaluated in 172 carriers of chromosomal abnormalities (CA) such as Down's syndrome (DS), Robertsonian translocations (RT), Klinefelter's and Turner's syndromes, trisomy Х, disomy Y, and various structural CA. In controls (n=318), AcRG dosage varied from 119 to 190 copies with a mean of 151 copies per diploid genome. In CA carriers, except for DS newborns, AcRG dosage was not beyond these limits. As shown previously, only within these limits cellular homeostasis and organism's viability can be supported, while genomes beyond these limits are eliminated by embryonic loss. About 10% of embryos with DS and 50% of embryos with RT die/are aborted exclusively due to a surplus (DS) or a shortage (RT) of AcRG. AcRG dosage also affects the CA carrier's viability after birth, as demonstrated by comparing newborn and aged (10-40 y.o.) DS patients. Sampling range of AcRG dosage becomes considerably narrower with age: DS newborns ranged from 139 to 194 RG copies (σ2=3.59), while aged DS patients varied from 152 to 190 copies (σ2=1.55) with the same mean. Each CA group showed peculiarities in AcRG dosage distribution. We found that carriers of numerical abnormalities of gonosomes (sex chromosomes) concentrate within the area of medium, most adaptive dosages, whilst carriers of structural CA can only survive with relatively high AcRG number. Our article is the first ever to report an association of CA viability with the genomic number of AcRG.

There are numerous applications of quantitative PCR for both diagnostic and basic research. As in many other techniques the basis of quantification is that comparisons are made between different (unknown and known or reference) specimens of the same entity. When the aim is to compare real quantities of different species in samples, one cannot escape their separate precise absolute quantification. We have established a simple and reliable method for this purpose (Ct shift method) which combines the absolute and the relative approach. It requires a plasmid standard containing both sequences of amplicons to be compared (e.g. the target of interest and the endogenous control). It can serve as a reference sample with equal copies of templates for both targets. Using the ΔΔCt formula we can quantify the exact ratio of the two templates in each unknown sample. The Ct shift method has been successfully applied for transposon gene copy measurements, as well as for comparison of different mRNAs in cDNA samples. This study provides the proof of concept and introduces some potential applications of the method; the absolute nature of results even without the need for real reference samples can contribute to the universality of the method and comparability of different studies.

Human pyruvate dehydrogenase complex (PDC) catalyzes a key step in the generation of cellular energy and is composed by three catalytic elements (E1, E2, E3), one structural subunit (E3-binding protein), and specific regulatory elements, phosphatases and kinases (PDKs, PDPs). The E1α subunit exists as two isoforms encoded by different genes: PDHA1 located on Xp22.1 and expressed in somatic tissues, and the intronless PDHA2 located on chromosome 4 and only detected in human spermatocytes and spermatids. We report on a young adult female patient who has PDC deficiency associated with a compound heterozygosity in PDHX encoding the E3-binding protein. Additionally, in the patient and in all members of her immediate family, a full-length testis-specific PDHA2 mRNA and a 5′UTR-truncated PDHA1 mRNA were detected in circulating lymphocytes and cultured fibroblasts, being bothmRNAs translated into full-length PDHA2 and PDHA1 proteins, resulting in the co-existence of both PDHA isoforms in somatic cells.Moreover, we observed that DNA hypomethylation of a CpG island in the coding region of PDHA2 gene is associatedwith the somatic activation of this gene transcription in these individuals. This study represents the first natural model of the de-repression of the testis-specific PDHA2 gene in human somatic cells, and raises some questions related to the somatic activation of this gene as a potential therapeutic approach for most forms of PDC deficiency. This study was supported in part by grants from the Fundação para a Ciência e a Tecnologia (FCT), Portugal: SFRH/BD/31264/2006 awarded to Ana Pinheiro, POCI/SAU-MMO/57052/2004 awarded to Isabel Rivera, and PEst-OE/SAU/UI4013/2013.

Amelogenesis imperfecta (AI) is a clinically and genetically heterogeneous disorder of tooth development which is due to aberrant deposition or composition of enamel. Both syndromic and isolated forms exist; they may be inherited in an X-linked, autosomal recessive, or autosomal dominant manner. WDR72 is one of ten currently known genes for recessive isolated AI; nine WDR72 mutations affecting single nucleotides have been described to date. Based on whole exome sequencing in a large consanguineous AI pedigree, we obtained evidence for presence of a multi-exonic WDR72 deletion. A home-made multiplex ligation-dependent probe amplification assay was used to confirm the aberration, to narrow its extent, and to identify heterozygous carriers. Our study extends the mutational spectrum for WDR72 to include large deletions, and supports a relevance of the previously proposed loss-of-function mechanism. It also introduces an easy-to-use and highly sensitive tool for detecting WDR72 copy number alterations.

CC chemokine ligand 3 like-1 (CCL3L1) encodes for CCL3L1 protein, which is a human immunodeficiency virus (HIV) suppressive chemokine and a potent ligand of HIV CCR5 co-receptor. CCL3L1 exhibits variation in the gene copy number (CN) and could influence HIV susceptibility through gene dosage effect. The study aims to determine the distribution of CCL3L1 CN among HIV subjects of Malay, Chinese, and Indian ethnics in Malaysia and to evaluate the impact of CCL3L1 CN on susceptibility to HIV. This study involved 182 HIV patients who attended outpatient clinics of three hospitals in Malaysia and 150 non-HIV (control) subjects. Typing of CCL3L1 CN was conducted via multiplex paralogue ratio tests (PRTs), followed by validation of the CCL3L1 CN by microsatellite analyses. Both Malay and Indian HIV subjects had the CN mode of two, while the CN mode for the Chinese was four. The CCL3L1 gene CN was found to be strongly associated with ethnicity (p 0.001) with the diverse distribution of CCL3L1 CN between the Malay (range = 0-6), Chinese (range = 0-9), and Indian (range = 1-4) ethnic groups. CCL3L1 CN higher than and equal to the average was associated with reduced HIV susceptibility among the Malays (p 0.05). However, the negative results found for the Indian and Chinese need to be further analysed in a larger sample size.

Codon usage bias is an important genomic phenomenon, where highly expressed genes use optimal codons for smoother translation with high yield, facilitated by the cognate tRNAs. Here, we presented the tRNA co-adaptation index (co-AI) by correlating tRNA gene copy number and codon composition in Saccharomyces cerevisiae. We observed that this co-AI is positively correlated with protein abundance and translation rate. Considering nucleotide substitutions, co-AI influences synonymous substitutions more than gene expression and protein abundance, the most important determinants of evolutionary rate. Co-AI correlates positively with mRNA secondary structure stability and mRNA half-life, which may lead to protein accumulation under high co-AI. However, the highly expressed proteins encoded by high co-AI genes are assisted by molecular chaperones to attain their proper functional conformation and prevent accumulation.

Oncogenes can potentially cause uncontrolled cell growth, leading to cancer development, and these genes are normally mutated and over-expressed in tumor cells. Genomic alteration of oncogenes might result in oncogenesis and promotion of cancer progression. To date, researchers have focused mainly on the roles of oncogenes in particular cancers, but investigation of oncogenes with gain-of-function mutations in multiple cancer types are less represented in the literature. Furthermore, the effect of those gain-of-function are not validated in gene expression level. To meet this demand, we performed a systematic analysis of gene expression in oncogenes to identify the occurrence of gain-of-function mutations in pan-cancer. We identified 33,551 oncogenic mutations in 5000 samples. From our analysis, we identified three tissues with the highest frequency of gain-of-functional oncogenic mutations in hundreds of samples: breast (739 samples), central nervous system (646 samples) and large intestine (498 samples). By further counting the number of occurrences of oncogenes across cancer types, we identified a list cross-cancer mutational signatures of 99 oncogenes highly mutated in >400 samples in breast, central nervous system and large intestine samples. By further overlapping with gene expression data in the matched tumor samples, we further identified 1875 gain-of-functional mutations/events with consistent gene up-regulation in 1031 samples from multiple cancers. This result may offer additional insight into the relationship between gene dosage and oncogenesis and maybe useful in targeted cancer therapy. In summary, this study provides the first globally examining on the genetic alteration of oncogenes across cancer types. Clinical association analysis has shown that these 99 genes have a significant effect on survival.

Cancer cells undergo a variety of DNA copy number gains and losses (CNV), raising two important questions related to cancer development: (i) Which genes are affected? (ii) And how do CNVs, that do not represent complete deletions but do represent gene-dosage alterations, impact cancer cell functions? Recent studies have indicated that CNVs in cancer can impact genes for regulatory proteins long known to be associated with cancer development, but less is understood about CNVs affecting effector genes. Also, we have recently indicated the likely importance of transcription factor binding site (TFBS) copies in effector genes, in regulating the transition from a proliferative to an apoptotic state. Here we report data-mining analyses that indicate that copies of apoptosis-effector genes are commonly lost in cancer development, in comparison to proliferation-effector genes, and when not, apoptosis effector genes have silenced chromatin structures.

The DEFB103 gene is a member of the β-defensin gene family. In this study, we applied multiple sets of primers to characterize the DEFB103 transcript. RT-PCR was used to determine the cDNA boundaries and it indicated that the cDNA start point is at least 514 bp before the start codon and not further than 678 bp. In addition, the length of the 3'UTR was determined to be at least 53 bp after the stop codon. Seven SNPs were located in the 5'UTR, and comprised 4 different haplotypes in genomic DNA. Using these haplotype data, it could be proven that at least two complete copies of DEFB103 with an ATG start codon are present in cDNA in most cattle. Additionally haplotype data indicated that there are also multiple incomplete copies in most cattle. A non-coding exon 1a, and a 261 bp intron 1a were identified in cattle, and subsequently predicted in sheep and goats. DEFB103 sequence assemblies and partial cloning sequences revealed two types of deletion (4 bp and 8 bp) in the 5'UTR. These observations could prove that these copies are not assembly artifact.

Interstitial duplications involving chromosome 11q have rarely been reported in the literature and mainly represent large, cytogenetically detectable rearrangements associated with a wide and variable spectrum of neurodevelopmental disorders. We report on a patient affected by intellectual disability, craniosynostosis, and microcephaly. Array-CGH analysis identified a de novo 290 kb interstitial duplication of chromosome 11q13.3 including the FGF3 and FGF4 genes. Clinical comparison of our patient with those previously reported with overlapping 11q duplications allows us to define the minimal duplicated region associated with craniosynostosis and strongly supports the hypothesis that the constitutional increased dosage of the FGF3 and FGF4 genes is a risk factor for craniosynostosis in humans.

Prolyl oligopeptidase (POP) is a widely distributed multifunctional protein which has an endopeptidase activity to cleave a -Pro-X- peptide bond. In spite of numerous studies about POP, the mechanism by which its transcription is controlled has not been well investigated. Here we generated transgenic mice bearing a transgene which contained a 914-bp POP gene promoter linked to the enhanced green fluorescent protein (EGFP) gene to assess the in vivo promoter activity. We established six transgenic lines with different copy numbers, but no EGFP signal was observed in four lines due to a high level of DNA methylation, which suggested that the transgene was subject to position effect. However, in the other two lines, we detected the EGFP expression in many tissues, and its placental localization showed a similar change to POP. A strong EGFP signal was observed in the junctional and labyrinthine zones of E10.5-E12.5 placentas and in the junctional zone and the maternal decidua after that. This placental gene activation might be attributed to AP-2 gamma because we detected its binding to the POP promoter. In contrast, we did not obtain any evidence that EGFP was expressed in a similar pattern compared with POP in the ovary. The current data demonstrated that the 914-bp promoter had sufficient activity to reproduce the POP localization in the placenta if it was not subject to position effect and suggest that the regulatory mechanism of the POP gene expression differs between tissues.(C) 2013 Elsevier B.V. All rights reserved.

Human mesenchymal stem cells (hMSCs) have attracted much attention for tissue repair and wound healing because of their self-renewal capacity and multipotentiality. In order to mediate an effective therapy, substantial numbers of cells are required, which necessitates extensive sub-culturing and expansion of hMSCs. Throughout ex vivo expansion, the cells undergo telomere shortening, and critically short telomeres can trigger loss of cell viability. Telomeres are nucleoprotein structures that cap the ends of chromosomes, and serve to protect the DNA from the degradation which occurs due to the end-replication problem in all eukaryotes. As hMSCs have only a finite ability for self-renewal like most somatic cells, assaying for telomere length in hMSCs provides critical information on the replicative capacity of the cells, an important criterion in the selection of hMSCs for therapy. Telomere length is generally quantified by Southern blotting and fluorescence in situ hybridization, and more recently by PCR-based methods. Here we describe the quantification of hMSC telomere length by real-time PCR; our results demonstrate the effect of telomere shortening on the proliferation and clonogenicity of hMSCs. Thus, this assay constitutes a useful tool for the determination of relative telomere length in hMSCs.

Down syndrome (DS) is the most common genetic aberration leading to intellectual disability. DS results from an extra copy of the long arm of human chromosome 21 (HSA21) and the increased expression of trisomic genes due to gene dosage. While expression in DS and DS models has been studied extensively at the RNA level, much less is known about expression of trisomic genes at the protein level. We have used quantitative Western blotting with antibodies to 20 proteins encoded by HSA21 to assess trisomic protein expression in lymphoblastoid cell lines (LCLs) from patients with DS and in brains from two mouse models of DS. These antibodies have recently become available and the 20 proteins largely have not been investigated previously for their potential contributions to the phenotypic features of DS. Twelve proteins had detectable expression in LCLs and three, CCT8, MX1 and PWP2, showed elevated levels in LCLs derived from patients with DS compared with controls. Antibodies against 15 proteins detected bands of appropriate sizes in lysates from mouse brain cortex. Genes for 12 of these proteins are trisomic in the Tc1 mouse model of DS, but only SIM2 and ZNF295 showed elevated expression in Tc1 cortex when compared with controls. Genes for eight of the 15 proteins are trisomic in the Ts65Dn mouse model of DS, but only ZNF294 was over expressed in cortex. Comparison of trisomic gene expression at the protein level with previous reports at the mRNA level showed many inconsistencies. These may be caused by natural inter-individual variability, differences in the age of mice analyzed, or post-transcriptional regulation of gene dosage effects. These antibodies provide resources for further investigation of the molecular basis of intellectual disability in DS.

LIM domains are zinc-binding motifs that mediate protein–protein interactions and are found in a wide variety of cytoplasmic and nuclear proteins. The nuclear LIM domain family members have a number of different functions including transcription factors, gene regulation, cell fate determination, organization of the cytoskeleton and tumour formation exerting their function through various LIM domain interacting protein partners/cofactors. Nuclear LIM domain interacting proteins/factors have not been reported in any protozoan parasites including Leishmania. Here, we report for the first time cloning, characterization and subcellular localization of nuclear LIM interactor-interacting factor (NLI) like protein from Leishmania donovani, the causative agent of Indian Kala-azar. Primary sequence analysis of LdNLI revealed presence of characteristic features of nuclear LIM interactor-interacting factor. However, leishmanial NLI represents a distinct kinetoplastid group, clustered in a separate branch of the phylogenic tree. The sub-cellular distribution of LdNLI revealed the discreet localization in nucleus and kinetoplast only, suggesting that the gene may have a role in parasite gene expression.

Made available in DSpace on 2018-11-27T21:47:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-11-30 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Secretaria de Ciencia e Tecnologia do Estado do Parana (SETI) Fundacao Araucaria de Apoio ao Desenvolvimento Cientifico do Estado do Parana (Fundacao Araucaria) Transposable elements are able to move along eukaryotic genomes. They are divided into two classes according to their transposition intermediate: RNA (class I or retrotransposons) or DNA (class II or DNA transposons). Most of these sequences are inactive or non-autonomous in eukaryotic genomes. Inactivate transposons can accumulate mutations at neutral rates until losing their molecular identity. They may either be eliminated from the genome or take on different molecular functions. Transposable elements may also participate in the differentiation of sex chromosomes. Therefore, the structural variations and nucleotide similarity of Tc1/Mariner sequences were analyzed along with their potential participation in the differentiation processes of sex chromosomes in the genomes of Parodontidae fish. All Parodontidae species presented non-autonomous copies of Tc1/Mariner with structural variation, different levels of deterioration (genetic distance), and variations in insertion and deletion patterns. The physical mapping of Tc1/Mariner on chromosomes revealed dispersed signals in euchromatins, with small accumulations in terminal regions and in the sex chromosomes. The gene dosage ratios indicated copy number variations of Tc1/Mariner among the genomes and high transposase open reading frame deterioration in Parodon hilarii and Parodon pongoensis genomes. This transposon presented transcriptional activity in gonads, but there was no significant difference between sexes. This may indicate non-functional protein expression or may correspond to DNA binding proteins derived from Tc1/Mariner. Thus, our results show Tc1/Mariner inactivation along with a diversity in Parodontidae genomes and its participation in the differentiation of the W sex chromosome. (C) 2016 Elsevier B.V. All rights reserved. Univ Fed Parana, Dept Genet, Curitiba, Parana, Brazil Univ Estadual Ponta Grossa, Dept Struct & Mol Biol & Genet, Ponta Grossa, Parana, Brazil Sao Paulo State Univ, Dept Bioproc & Biotechnol, Botucatu, SP, Brazil Sao Paulo State Univ, Inst Biosci, Dept Morphol, Sao Paulo, Brazil Univ Fed Sao Carlos, Dept Genet & Evolut, Sao Carlos, SP, Brazil Sao Paulo State Univ, Dept Bioproc & Biotechnol, Botucatu, SP, Brazil Sao Paulo State Univ, Inst Biosci, Dept Morphol, Sao Paulo, Brazil CNPq: 303696/2013-3 CNPq: 473212/2013-7 CAPES: 40001016006 FAPESP: 2012/15258-0 FAPESP: 2013/04533-3 Secretaria de Ciencia e Tecnologia do Estado do Parana (SETI): 378/2012 Fundacao Araucaria de Apoio ao Desenvolvimento Cientifico do Estado do Parana (Fundacao Araucaria): 22.843

Objective Individuals with chromosomal aneuploidies tend to develop malignancies. Telomerase is an enzyme complex that lengthens telomeres and has enhanced expression in numerous malignancies; one of its components is encoded by the TERC gene. In this study, we evaluated the TERC gene copy number in amniocytes from fetuses with aneuploidy, other than trisomy-21. Methods In this prospective, basic research study, fluorescence in situ hybridization (FISH) for the TERC gene (3q26) was applied to amniocytes retrieved from 14 fetuses with various aneuploidies and from a control group of 6 fetuses with a normal karyotype, to determine the TERC gene copy number. Results The percentage of cells with more than two copies of the TERC gene was lowest in the control group (x3 = 1.2 ± 0.4%; x4 = 0 ± 0%), higher in the sex chromosome aneuploidies (x3 = 4 ± 3%; x4 = 0.7 ± 0.95%) and even higher in trisomy 18 (x3 = 10.6 ± 2.3; x4 = 4.6 ± 1.8). The differences were statistically significant (P Conclusion The TERC gene copy number is increased in aneuploid amniocytes, which demonstrates their genetic instability and is presumably related to their tendency to develop malignancies.

We investigated the genetic defects in two families with classic congenital adrenal hyperplasia (CAH). Clinical data and vein blood of the family members were collected, hormonal evaluation, karyotype analysis, ultrasound and CT scans were performed, and a direct-sequencing of PCR products of the candidate genes was used to identify the mutations. In family A, Patients A-II:1 and A-II:2 were found to be in a compound heterozygous state for mutations of p.I172N (g.1004 T > A) in exon 4 and IVS2-13A/C > G (g.659A/C > G) in intron 2 in CYP21A2 gene, their father A-I:1 and mother A-I:2 were found to carry a heterozygous mutation of IVS2-13A/C > G (g.659A/C > G) and p.I172N (g.1004 T > A) respectively. In family B, Patient B-II:1 was detected to carry only one heterozygous mutation of p.I172N; no other mutations in CYP11B1, CYP17A1 or HSD3B2 genes were detected. Her father B-I:1 carried a heterozygous mutation of p.I172N (g.1004 T > A) and her mother B-I:2 was found to be a wild type in all the candidate genes. Obviously, patients A-II:1 and A-II:2 inherited the p.I172N (g.1004 T > A)-bearing maternal allele and the IVS2-13A/C > G (g.659A/C > G)-bearing paternal allele. And this kind of compound heterozygous mutations caused simple virilising (SV) CAH. While patient B-II:1, whose phenotype was SV CAH too, was found to carry only one heterozygous mutation of the p.I172N (g.1004 T > A)-bearing paternal allele, and needed further studies.

Genes must be stably integrated into bacterial chromosomes for complementation of gene deletion mutants in animal infection experiments or to express antigens in vaccine strains. However, with currently available vectors it is cumbersome to create multiple, stable, unmarked chromosomal integrations in mycobacteria. Here, we have constructed a novel integration vector for mycobacteria that enables expression of genes from a cassette protected from transcriptional interference by bi-directional transcriptional terminators proven to be highly efficient in in vitro transcription termination assays. Removal of the integrase gene by a site-specific recombinase, easily identifiable by loss of a backbone reporter gene, stabilizes the integration cassette and makes this vector ideally suitable for infection experiments. This integration vector can be easily adapted to different mycobacteriophage attachment sites (attB) due to its modular design. Integration of a gfp expression cassette at the L5, Giles and Ms6 attB sites in the chromosomes of Mycobacterium smegmatis and Mycobacterium tuberculosis yielded identical gfp expression levels, indicating that none of these sites are compromised for gene expression. The copy number of pAL5000-based extrachromosomal plasmids is 23 in M. smegmatis as determined by quantitative real-time PCR and accounts for the previously observed drastic reduction of gene expression upon integration of plasmids into the chromosome of mycobacteria. Gfp expression and fluorescence of M. smegmatis and M. tuberculosis strains with multiple integrations of gfp increased concomitantly with the copy number demonstrating that these vectors can be used to generate stronger phenotypes and/or to analyze several genes simultaneously in vivo.

Allomyces arbuscula, a primitive chytridiomycete fungus, has two Ca 2+ -dependent cysteine proteases, the CDP I and CDP II. We have cloned and analyzed the nucleotide sequence of CDP II gene and domain structure of the protein. Blast analysis of the sequence has shown that the protein belongs to a newly described member of caspase superfamily protein, the metacaspase, a CD clan of C14 family cysteine protease, we hence-forth name it as AMca 2 ( Allomyces metacaspase 2). Southern hybridization studies have shown that the gene exists in a single copy per genome. The transcriptional analysis by Northern hybridization has confirmed our previous results that the protein is developmentally regulated, i.e. present in active growth phase but disappears during nutritional stress which also induces reproductive differentiation, indicating that the protein promotes cell growth, not death. The recombinant gene product expressed in E scherichia coli has all the catalytic properties of native enzyme, i.e. sensitivity to protease inhibitors and substrate specificity. There is an absolute requirement of Ca 2+ for the activation of catalytic activity and the presence of R residue at the cleavage site (P1 position) in the substrate. The presence of a second basic residue, either R or K, in the P2 position strongly inhibits the catalytic activity which is stimulated by the presence of P and to a lesser extent G at this site. Peptide substrates with D at the cleavage site are not recognised and therefore not cleaved. The enzyme activity is inhibited by EDTA–EGTA, cysteine protease inhibitors and a specific peptide inhibitor Ac GVRCHCL TFA, but not by E64, although a potent inhibitor of cysteine proteases.

The paralogous genes RABL2A (chr2) and RABL2B (chr22) emerged by duplication of a single gene in the human–chimpanzee ancestor and share a high degree of sequence similarity. In Phelan–McDermid-Syndrome microdeletions of 22q13 often also affecting RABL2B are of clinical importance but their incidence is still unknown. We analyzed a German population (190 individuals) for such aneuploidies and the paralogs’ expression in cell lines by RABL2 paralogous sequence quantification. For determination of the genomic and transcriptional ratios of RABL2A and RABL2B a Pyrosequencing protocol was introduced as a high-throughput method. During PCR the 3′ end of the biotinylated strand is engineered by a backfolding oligonucleotide to hybridize in the Pyrosequencing reaction to an internal site near the sequence to be analyzed. In human samples no deviations of the euploid genomic state could be detected indicating that 22q13 microdeletions involving RABL2B are rare. However, despite equal gene dosage a preferential expression of RABL2B in human tissues and lymphoblastoid cell lines was detected which is most pronounced in brain and placenta. This renders a complete functional complementation of one paralog by the respective other unlikely and hints to a functional and clinical importance, in particular with respect to the 22q13 chromosomal deletion syndrome. Remarkably and in contrast to human, expression levels of the two paralogs in a chimpanzee cell line are equal. This finding is discussed in view of the relocation of RABL2A from its ancestral telomeric to its pericentromeric location in human.

Miniature inverted-repeat transposable elements (MITEs) are small and high copy number transposons, related to and mobilized by some class II autonomous elements. New MITE families can be identified by computer-based mining of sequenced genomes. We describe four MITE families related to MtPH transposons mined de novo in the genome of Medicago truncatula, together with one previously described family MITRAV. Different levels of their intra-family sequence diversity and insertion polymorphism indicate that they were active at different evolutionary periods. MetMIT1 and MITRAV families were uniform in sequence and produced highly polymorphic insertion sites in 26 ecotypes representing a M. truncatula core collection. A subset of insertions was present only in the reference genome of A17 ‘Jemalong’, suggesting that the two families might have been active in the course of domestication. In contrast, all investigated insertions of the MetMIT2 family were fixed, showing that it was not active after M. truncatula speciation. MetMIT1 elements were divided into three clusters, i.e. (I) relatively heterogenous copies fixed in the genome of M. truncatula, (II) uniform but also mostly fixed, and (III) uniform and polymorphic among the investigated accessions. It might reflect the evolutionary history of the MetMIT1 family, showing multiple bursts of activity. A number of MetMIT1 and MITRAV insertions were present within 1 kb upstream or downstream the ORF. A high proportion of insertions proximal to coding regions was unique to A17 ‘Jemalong’.

Cdc28 is the main cyclin-dependent kinase (CDK) directing the cell cycle in the budding yeast Saccharomyces cerevisiae. Besides cyclin binding, Cdc28 requires phosphorylation by the Cak1 kinase to achieve full activity. We have previously isolated carboxy-terminal cdc28(CST) mutants that are temperature sensitive and exhibit high chromosome instability. Both phenotypes are suppressed by high copy Cak1 in a manner that is independent of its catalytic activity and conversely, combination of cdc28(CST) and cak1 mutations results in synthetic lethality. Altogether, these results suggest that for the Cdc28 complexes to remain stable and active, an interaction with Cak1 is needed via the carboxyl terminus of Cdc28. We report two-hybrid assay data that support this model, and results that indicate that actively growing yeast cells require an optimum Cdc28:Cak1 ratio. While Cak1 is constitutively active and expressed, dividing cells tightly regulate Cak1 protein levels to ensure presence of adequate levels of Cdc28 CDK activity.

A novel gene encoding a DEAD-box RNA helicase designated as GmRH was isolated from soybean. Amino acid sequence alignment and phylogenetic tree analysis revealed a close relationship between GmRH and other orthologous DEAD-box RNA helicases from other plant species. Structural motif analysis revealed that the bipartite lysine rich nuclear localization signal (NLS) is present in the N-terminal variable region of GmRH and that there are ten conserved motifs found in DEAD-box RNA helicase proteins. Southern blot analysis revealed the presence of 2 copies of GmRH in the soybean genome. Northern blot analysis demonstrated that the RNA expression of the GmRH was induced during low temperature or high salinity stress, but not by the exogenous application of abscisic acid or drought stress. Subcellular localization studies showed that GmRH((1-355))-GFP is localized in the nucleus, whereas GmRH((130-355))-GFP is localized both in the cytoplasm and in the nucleus. This provides the evidence that the N-terminal region predicted as NLS is essential for nuclear targeting of the GmRH protein in the plant cell. Purified GST-GmRH recombinant protein was shown to unwind dsRNA independent of ATP in vitro. Here, we propose that GmRH plays an important role in RNA processing during low temperature and high salinity stresses in plants.

Fish odorant receptors comprise several distinct families of G protein-coupled receptors. The repertoires of fish vomeronasal receptor family 2 (V2R) genes differ substantially among species, which seems to be related to the different odor sensitivity among fish species. In this study, the number and diversity of V2R genes in a cyprinid fish, bitterling Tanakia lanceolata, were examined by gene cloning and genomic Southern blot analysis. Fifty-six distinct V2R partial sequences were identified by extensive cloning of degenerate PCR fragments. Phylogenetic analysis of the V2R genes in bitterling and other model fishes showed that the bitterling V2Rs were subdivided into eight subfamilies that diverged before the separation of cyprinids and other fishes. Most bitterling V2Rs belonged to two major subfamilies, while only one or a few genes were identified in the remaining six subfamilies. The repertoire of bitterling V2Rs was quite similar to that of zebrafish, although some bitterling-specific gene expansions have also occurred. Based on the genomic Southern blot analysis, the copy number of the bitterling V2R gene was estimated at least 60, similar to the number of V2R genes found in the zebrafish genome. This also suggests that bitterling has relatively larger number of V2R genes than other model fishes.

Genetic mutations can occur on a wide variety of scales, including those that change single nucleotides, those that add or remove content to/from a genome, and those that change the organization of a genome. Gene duplications are a specific class of mutations that add content to a genome, and they can arise via a wide variety of mechanisms. I examined the mechanisms responsible for recently duplicated genes in the D. pseudoobscura genome, and I observed both retroposed and DNA duplications. Many duplicated genes lack signatures of either retroposition or DNA-based mechanisms, but other features of these ambiguously duplicated genes suggest that most were generated via retroposition. Furthermore, close examination of sequences flanking DNA duplications and those found at the breakpoints of chromosomal inversions suggests a connection between these two events. In Drosophila, duplicated genes near inversion breakpoints can arise via unequal genetic exchange during the non-allelic crossing over event giving rise to the inversion. I observed one duplicated gene in the D. pseudoobscura genome that appears to have been generated by this mechanism. Additionally, many DNA duplications in the D. pseudoobscura genome are flanked by a repetitive sequence also found at the breakpoints of chromosomal inversions. This suggests that the molecular mechanisms responsible for chromosomal rearrangements and some duplicated genes have overlapping processes.

Transposable elements (TE) are functionally important genetic elements that can move within a genome. Miniature inverted-repeat transposable elements (MITEs) constitute a class of transposable elements that are usually small in size and have high numbers of conserved copies. Identifying all the MITEs in a genome could provide new insights about gene evolution and genome dynamics of the organism. We present a web-based MITE Uncovering SysTem (MUST) for prediction and analyses of MITEs at a genome level. MUST reliably found both the previously known and novel MITEs in the two bacterial genomes, Anabaena variabilis ATCC 29413 and Haloquadratum walsbyi DSM 16790. MUST is available at http://csbl1.bmb.uga.edu/ffzhou/MUST/ (the standalone version is available upon request). Supplementary data associated with this article are available in the online version or at: http://csbl1.bmb.uga.edu/ffzhou/MUST/supp/.

Gene duplication and alternative splicing (AS) are the two major evolutionary mechanisms that can bring the functional variation by increasing gene diversification. The purpose of this research is to understand the evolutionary relationship between these two different mechanisms, utilizing available data resources. We found the proportion of AS loci and the average number of AS isoforms per locus to be larger in duplicated genes compared to those in singleton genes. However we also found that small gene families have larger proportion of AS loci and larger average number of AS isoforms per locus than large gene families. These results suggest that gene duplication allows for more alternative splicing events to occur on newly duplicated copies than on singletons, probably due to the reduced functional constraint on the duplicates. Smaller average number of AS isoforms in the larger gene families can be explained by the decreased possibility for new useful function to be created via a new alternative splicing event.

The melanocortin type 1 receptor (Mc1r) belongs to a family of G-protein-coupled receptors involved in various physiological processes in vertebrates. Melanocortins, the Mcr natural agonists, are pituitary peptide hormones including adrenocorticotropin and melanocyte-stimulating hormones. In mammals and birds, Mc1r is involved in pigmentation and expressed in melanocytes and melanoma. Activation of Mc1r leads to eumelanin production as well as to proliferation and survival of melanocytes in the epidermis. Here we report the molecular and evolutionary analysis of mc1r from three major fish models, the zebrafish Danio rerio, the medaka Oryzias latipes and the platyfish Xiphophorus maculatus. In contrast to some other melanocortin receptor genes, mc1r has been conserved as a single copy gene in divergent fish species. Its expression was detected in all organs tested in platyfish and medaka but was restricted to eyes, skin, brain and testis in zebrafish, this possibly reflecting differences in the distribution of extracutaneous melanophores. The mc1r gene was found to be expressed during embryogenesis, as well as in Xiphophorus hybrid melanoma, similar to human tumours. Protein sequence comparison between fish and mammalian Mc1r revealed a remarkable concordance between evolutionary and functional analyses for the identification of residues and regions critical for receptor function.

Synonymous codon usage and cellular tRNA abundance are thought to be co-evolved in optimizing translational efficiencies in highly expressed genes. Here in this communication by taking the advantage of publicly available gene expression data of rice and Arabidopsis we demonstrated that tRNA gene copy number is not the only driving force favoring translational selection in all highly expressed genes of rice. We found that forces favoring translational selection differ between GC-rich and GC-poor classes of genes. Supporting our results we also showed that, in highly expressed genes of GC-poor class there is a perfect correspondence between majority of preferred codons and tRNA gene copy number that confers translational efficiencies to this group of genes. However, tRNA gene copy number is not fully consistent with models of translational selection in GC-rich group of genes, where constraints on mRNA secondary structure play a role to optimize codon usage in highly expressed genes.

Distal control of the whey acidic protein (WAP) locus was studied using a transgenic approach. A series of pig genomic fragments encompassing increasing DNA lengths upstream of the mammary specific whey acidic protein (WAP) gene transcription start point (tsp) and 5 kb downstream were used for microinjection in mouse fertilized eggs. Our data pointed out three regions as potent regulators for WAP but not for RAMP3 gene expression (a non mammary-specific gene located 30 kb upstream of the WAP gene). WAP gene activating elements were present in the -80 kb to -30 kb and -145 kb to -130 kb regions whereas inhibitors were present in the -130 kb to -80 kb region. The stimulatory regions were characterized by peaks of histone H4 acetylation and a poor nucleosome occupancy in lactating sow mammary glands but not in liver. These data reveal for the first time the existence of several remote potent regulatory regions of the pig WAP gene.

Mutation in the inward rectifier potassium channel gene, kir7.1, was previously identified as being responsible for the broader stripe zebrafish skin pattern mutant, jaguar/obelix. An amino acid substitution in this channel causes a broader stripe pattern than that of wild type zebrafish. In this study we analyzed cichlid homologs of the zebrafish kir7.1 gene. We identified two kinds of homologous genes in cichlids and named them cikir7.1 and cikir7.2. Southern hybridization using cichlid genome revealed that cichlids from the African Great Lakes, South America and Madagascar have two copies of the gene. Cichlids from Sri Lanka, however, showed only one band in this experiment. Database analysis revealed that only one copy of the kir7.1 gene exists in the genomes of the teleosts zebrafish, tetraodon, takifugu, medaka and stickleback. The deduced amino acid sequence of cikir7.1 is highly conserved among African cichlids, whereas that of cikir7.2 has several amino acid substitutions even in conserved transmembrane domains. Gene expression analysis revealed that cikir7.1 is expressed specifically in brain and eye, and cikir7.2 in testis and ovary; zebrafish kir7.1, however, is expressed in brain, eye, skin, caudal fin, testis and ovary. These results suggest that gene duplication of the cichlid kir7.1 occurred in a common ancestor of the family Cichlidae, that the function of parental kir7.1 was then divided into two genes, cikir7.1 and cikir7.2, and that the evolutionary rate of cikir7.2 might have been accelerated, thereby effecting functional diversification in the cichlid lineage. Thus, the evolution of kir7.1 genes in cichlids provides a typical example of gene duplication—one gene is conserved while the other becomes specialized for a novel function.

A new Escherichia coli host/vector system has been developed to allow a dual regulation of both the plasmid copy number and gene expression. The new pN15E vectors are low copy number plasmids based on the replicon of temperate phage N15, comprising the repA replicase gene and cB repressor gene, controlling the plasmid copy number. Regulation of pN15E copy number is achieved through arabinose-inducible expression of phage N15 antirepressor protein, AntA, whose gene was integrated into the chromosome of the host strain under control of the PBAD promoter. The host strain also carried phage N15 partition operon, sop, allowing stable inheritance of pN15E vectors in the absence of selection pressure. In the first vector, pN15E4, the same PBAD promoter controls expression of a cloned gene. The second vector, pN15E6, carries the phage T5 promoter with a double lac operator repression module thus allowing independent regulation of promoter activity and copy number. Using the lacZ gene to monitor expression in these vectors, we show that the ratio of induction/repression can be about 7600-fold for pN15E4 and more than 15,000-fold for pN15E6. The low copy number of these vectors ensures very low basal level of expression allowing cloning genes encoding toxic products that was demonstrated by the stable maintenance of a gene encoding a restriction endonuclease in pN15E4. The tight control of transcription and the potential to regulate gene activities quantitatively over wide ranges will open up new approaches in the study of gene function in vivo and controlled expression of heterologous genes.

Despite a wide distribution of transposable elements (TEs) in the genomes of higher eukaryotes, much of their evolutionary significance remains unclear. Recent studies have indicated that TEs are involved with biological processes such as gene regulation and the generation of new exons in mammals. In addition, the completion of the genome sequencings in Arabidopsis thaliana and Oryza sativa has permitted scientist to describe a genome-wide overview in plants. In this study, we examined the positions of TEs in the genome of O. sativa. Although we found that more than 10% of the structural genes contained TEs, they were underrepresented in exons compared with non-exonic regions. TEs also appeared to be inserted preferentially in 3'-untranslated regions in exons. These results suggested that purifying selection against TE insertion has played a major role during evolution. Moreover, our comparison of the numbers of TEs in the protein-coding regions between single copy genes and duplicate genes showed that TEs were more frequent in duplicate than single copy genes. This observation indicated that gene duplication events created a large number of functionally redundant genes. Subsequently, many of them were destroyed by TEs because the redundant copies were released from purifying selection. Another biological role of TEs was found to be the recruitment of new exons. We found that approximately 2% of protein-coding genes contained TEs in their coding regions. Insertion of TEs in genic regions may have the potential to be an evolutionary driving force for the creation of new biological functions.

The Mutator system of maize has been described as the most active and mutagenic plant transposon. The autonomous element MuDR contains two genes: mudrA encoding the transposase and mudrB whose product function remains undetermined. MudrA-like coding domain showed to be the most abundantly expressed transposon-related sequence in sugarcane transcriptome. A previous report identified the existence of at least four clades of mudrA-like sequences in sugarcane, rice and arabidopsis, which already existed prior to the Monocot-Eudicot split. To gain understanding about the abundance, distribution, copy number and diversity of mudrA-like sequences, a comparative study between sugarcane and rice was performed. As a result, it was possible to identify that copy number greatly differs and, at least in grasses, there was a class-specific amplification with a burst of Class II elements. Structural analyses performed on rice genomic sequences revealed that while Class I and Class II clades comprise elements with transposon features, Class III and Class IV no longer possess TIRs and correspond to domesticated transposases.

Reaumuria soongorica is one of the typical desert plants that present excellent tolerance to adverse environments. However, its molecular response to UV-B radiation remains poorly understood. To test the response and tolerance mechanisms of R. soongorica to the increasing UV-B radiation, the differentially expressed genes (DEGs) were investigated between the control and UV-B radiation groups. A total of 2150 DEGs were detected between the two groups, of which 561 were up-regulated and 1589 were down-regulated. For functional analysis, DEGs were divided into three groups: (i) Chloroplast-localized proteins, including photosynthesis-associated proteins, ribulose-phosphate-3-epimerase, and ATP-dependent Clp protease. Their transcripts were inhibited, implying that the normal function of chloroplast was affected by UV-B radiation. (ii) Proteins involved in signaling transduction, such as phototropins and GTP-binding proteins. The transcriptional alternation of phototropins may reduce the penetration of UV-B radiation by regulating phototropism, stomatal opening, and chloroplast relocation. The down regulation of GTP-binding proteins may inhibit replication of potentially damaged DNA through preventing cell division; and (iii) proteins for lipid transfer and flavonoids biosynthesis. The up-regulation of these genes suggested that lipid transfer and flavonoids may have a protective function in response to UV-B radiation. Thus, UV-B radiation may lead to the disruption of chloroplasts function. The induction of genes for signal transduction and protective proteins may be a strategy for responding to UV-B radiation in R. soongorica.