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PubMed1986

Regulation of herpes simplex virus 1 genes: alpha gene sequence requirements for transient induction of indicator genes regulated by beta or late (gamma 2) promoters.

This laboratory reported earlier that chimeric genes consisting of the structural sequences of the thymidine kinase (TK) gene fused to the promoter-regulatory domains of late (gamma 2) genes were regulated as bonafide gamma 2 genes when resident in the herpes simplex virus 1 genome but could not be differentiated from beta genes when introduced by transfection and stably integrated into the environment of the host genome (S. Silver and B. Roizman, Mol. Cell. Biol. 5, 518-528, 1985). We report here that beta-TK and the chimeric gamma 2-TK gene transfected into TK- baby hamster kidney (BHKtk-) were induced by alpha 4 and alpha 0 but not by the other alpha genes. Specifically: Both TK genes were induced by cotransfection with DNA fragments carrying an intact alpha 4 or an intact alpha 0 gene, but not by fragments carrying only the promoter-regulatory domain or the structural sequences of the alpha 4 gene or intact alpha 22, alpha 27, and alpha 47 genes. An alpha 4 gene carrying a 2700-bp deletion in its 3' coding sequence also induced both genes, although less efficiently. RNA homologous to the alpha 4 gene recovered from the cytoplasm of cells transfected with either the intact or truncated alpha 4 gene mapped to the bonafide site of transcription initiation of the alpha 4 gene. RNA homologous to the chimeric TK gene extracted from the cytoplasm of cells transfected with both gamma 2-TK and the alpha 4 gene was transcribed from the bonafide gamma 2 gene capping site fused to the TK gene. These results indicate that the alpha 4 gene and the alpha 0 gene are each capable of inducing the expression of both beta and gamma 2 genes resident in the environment of the cellular genome, that the active site responsible for induction is located near the N terminus of the alpha 4 protein, and reinforce the conclusion that gamma 2 genes resident in the environment of the host cell cannot be used to identify the authentic determinants of gamma 2 gene regulation by currently available tests.

Animals↗

PubMed2006

Further understanding human disease genes by comparing with housekeeping genes and other genes.

BACKGROUND: Several studies have compared various features of heritable disease genes with other so called non-disease genes, but they have yielded some conflicting results. A potential problem in those studies is that the non-disease genes contained a large number of essential genes--genes which are indispensable for humans to survive and reproduce. Since a functional disruption of an essential gene has fatal consequences, it's more reasonable to regard essential genes as extremely severe "disease" genes. Here we perform a comparative study on the features of human essential, disease, and other genes. RESULTS: In the absence of a set of well defined human essential genes, we consider a set of 1,789 ubiquitously expressed human genes (UEHGs), also known as housekeeping genes, as an approximation. We demonstrate that UEHGs are very likely to contain a large proportion of essential genes. We show that the UEHGs, disease genes and other genes are different in their evolutionary conservation rates, DNA coding lengths, gene functions, etc. Our findings systematically confirm that disease genes have an intermediate essentiality which is less than housekeeping genes but greater than other human genes. CONCLUSION: The human genome may contain thousands of essential genes having features which differ significantly from disease and other genes. We propose to classify them as a unique group for comparisons of disease genes with non-disease genes. This new way of classification and comparison enables us to have a clearer understanding of disease genes.

Adolescent↗

PubMed1981

Regulation of alpha genes of herpes simplex virus: expression of chimeric genes produced by fusion of thymidine kinase with alpha gene promoters.

We report a system for investigating promoters of eucaryotic cell and virus genes based on analyses of the regulation of herpes simplex virus 1 (HSV-1) thymidine kinases whose structural gene sequences have been fused to the promoter of the gene under study. In infected cells, the polypeptides specified by HSV-1 form at least three groups, alpha, beta and gamma, whose synthesis is coordinately regulated and sequentially ordered at the transcriptional level. To identify the DNA sequence responsible for the regulation of transcription of alpha genes, we fused the sequence encoding the 5' end of an alpha gene to the structural gene sequence of the thymidine kinase, a beta gene. The resultant recombinant DNA was inserted into the viral genome and was also used to convert Ltk- cells to tk+ phenotype. In cells infected with recombinant virus, the thymidine kinase gene was regulated and expressed as an alpha gene-that is, it was transcribed and processed in the absence of prior infected cell protein synthesis. Moreover, mRNA selected by hybridization to sequences encoding the thymidine kinase contains at its 5' terminus sequences homologous to the donor sequence encoding the t'terminus of the alpha mRNA. In converted tk+ cells, the fused thymidine kinase gene, like the wild-type gene, is stimulated by superinfection with the tk- virus. However, the stimulation is many times greater and is due to non-alpha-gene products, whereas in cells converted by the wild-type gene, the stimulation is by alpha gene products. We conclude that the alpha genes are identified for transcription by sequences at or near those encoding the 5' terminus of the mRNA, and transposition of these sequences to a beta gene is all that is required to convert it to an alpha gene. Transcription of alpha genes appears to be regulated by non-alpha-gene products, which could be contained within the structure of the virion. In converted Ltk+ cells, the thymidine kinase gene uses its own promoter.

Base Sequence↗

PubMed1989

Only DFL16, DSP2, and DQ52 gene families exist in mouse immunoglobulin heavy chain diversity gene loci, of which DFL16 and DSP2 originate from the same primordial DH gene.

In mice, 12 germ-line DH genes belonging to three different families (DQ52, DSP2 and DFL16) have been identified. The DH genes other than DQ52 are clustered in the 60 kb-long region located between VH and JH genes. Since there are seven DH gene families (DHQ52, DXP, DA, DK, DN, DM and DLR) in humans, we tried to identify new DH gene families in the 60 kb-long region using human DH gene probes. Mouse and human DH genes showing the highest similarity were mouse DFL16 genes and human DA genes. Southern hybridization of the mouse clones covering the 60-kb region with human DH probes did not detect any other DH genes. Nucleotide sequence analysis of the 4.0-kb fragment containing the DFL16.1 gene confirmed this conclusion. Comparison of the 12 germ-line DH genes and more than 150 somatic DH sequences also indicated that there are not more germ-line DH genes in the mouse genome. Moreover, comparison of nucleotide sequences of DFL16.1 and DSP2.2 genes and their surrounding regions suggests that both DH gene families originate from the same primordial DH gene. Using the flanking sequences of both DH genes, the divergence date between DFL16 and DSP2 genes was estimated at around 37 million years ago.

Animals↗

PubMed1992

Molecular analysis of the Bacillus subtilis bacteriophage SPP1 region encompassing genes 1 to 6. The products of gene 1 and gene 2 are required for pac cleavage.

Packaging of Bacillus subtilis phage SPP1 DNA into viral capsids is initiated at a specific DNA site termed pac. Using an in vivo assay for pac cleavage, we show that initiation of DNA synthesis and DNA packaging are uncoupled. When the DNA products of pac cleavage were analyzed, we could detect the pac end that was destined to be packaged, but we failed to detect the other end of the cleavage reaction. SPP1 conditional lethal mutants, which map adjacent to pac, were analyzed with our assay. This revealed that the products of gene 1 and gene 2 are essential for pac cleavage. SPP1 mutants that are affected in the genes necessary for viral capsid formation (gene 41) or involved in headful cleavage (gene 6) remain proficient in pac site cleavage. Analysis of the nucleotide sequence (2.769 x 10(3) base-pairs) of the region of the genes required for pac cleavage revealed five presumptive genes. We have assigned gene 1 and gene 2 to two of these open reading frames (orf), giving the gene order gene 1-gene 2-orf 3-orf 4-orf 5. The direction of transcription of the gene 1 to orf 5 operon and the length of the mRNAs was determined. We have identified, upstream from gene 1, the major transcriptional start point (P1). Transcription originating from P1 requires a phage-encoded factor for activity. The organization of gene 1 and gene 2 of SPP1 resembles the organization of genes in the pac/cos region of different Escherichia coli double-stranded DNA phages. We propose that the conserved gene organization is representative of the packaging machinery of a primordial packaging system.

Amino Acid Sequence↗

PubMed1997

Sequence analysis of gene 3, gene 4 and gene 5 of avian infectious bronchitis virus strain CU-T2.

We have previously reported the nucleotide sequences of gene 2 (spike (S) protein gene), gene 6 (nucleocapsid (N) protein gene), and the 3' end untranslated region of a novel avian infectious bronchitis virus (IBV) strain, CU-T2 [Jia et al. (1995) Arch. Virol. 140, 259 271]. In the present report we describe the sequences of the remaining genes of this strain (gene 3, 4 and 5) with the exception of gene 1 (RNA polymerase gene). Gene 3 contained three open reading frames (ORFs), 3a, 3b and 3c of 174, 195 and 282 nucleotides (nt), respectively. Gene 4 (membrane (M) protein gene) consisted of 749 nt with a single ORF of 687 nt. Gene 5 contained two ORFs, 5a and 5b, with 198 and 249 nt, respectively. Thus, in total, there were 7349 nt from the 5' end of S protein gene to the 3' end of the CU-T2 genome. The overall nt sequence homologies between gene 3, 4, and 5 of CU-T2 and those of other strains were between 84.1-90.8%, 85.8-88.8% and 90.4 96.4%, respectively. The predicted amino acid (aa) sequence homologies revealed that gene 3b and 5b were more conserved than 3a, 3c and 5a. Each individual gene of CU-T2 strain (with the exception of the RNA polymerase gene) had a different level of homology with the homologous gene of other strains, suggesting that the evolution of IBV strains in general has been a complex, and as yet, poorly understood process.

Amino Acid Sequence↗

PubMed2006

PDGF gene therapy enhances expression of VEGF and bFGF genes and activates the NF-kappaB gene in signal pathways in ischemic flaps.

BACKGROUND: Gene therapy is a novel approach for enhancing the viability of ischemic flaps. Expression of growth factor genes pertinent to angiogenesis and activation of genes of relevant signal pathways are imperative for improving flap viability. The authors investigated the gene expression profiles of growth factors and signal transduction pathways in ischemic flaps after PDGF gene therapy. METHODS: Twenty Sprague-Dawley rats were divided into two groups. The experimental group (n = 10) received the plasmid vector containing the PDGF cDNA injected into the dermis of the flap area, whereas the control group (n = 10) received the physiologic saline. Seven days later, a dorsal random flap was raised. Seven days after surgery, flap viability was assessed, and expression of VEGF, bFGF, TGF-beta1, NF-kappaB, Erk2, Stat1, and Smad2 genes of the NF-kappaB, MAPK, JAK-STAT, and Smad pathways was assessed by quantitative analysis of the products of reverse-transcriptase polymerase chain reaction. RESULTS: Transfer of exogenous PDGF gene significantly improved flap viability (p = 0.011). Levels of expression of VEGF and bFGF genes in the flap were significantly elevated after PDGF gene transfer (p = 0.0001 and p = 0.001, respectively). Expression of the NF-kappaB gene was significantly elevated (p = 0.041). In contrast, expression of TGF-beta1, and Erk2, Stat1, and Smad2 genes was not changed. CONCLUSIONS: Transfer of exogenous PDGF gene to ischemic flaps promotes expression of VEGF and bFGF genes and activation of NF-kappaB gene in addition to its effects on the PDGF gene. The finding implies that transfer of the gene of one growth factor ultimately improves the expression of the genes of multiple growth factors. Activation of the NF-kappaB gene suggests that the NF-kappaB pathway may be important in enhancement of flap viability and will likely be a target of future efforts of regulation of signaling process in treatment of ischemic flaps.

Animals↗

PubMed2005

Elucidation of gene-to-gene and metabolite-to-gene networks in arabidopsis by integration of metabolomics and transcriptomics.

Since the completion of genome sequences of model organisms, functional identification of unknown genes has become a principal challenge in biology. Post-genomics sciences such as transcriptomics, proteomics, and metabolomics are expected to discover gene functions. This report outlines the elucidation of gene-to-gene and metabolite-to-gene networks via integration of metabolomics with transcriptomics and presents a strategy for the identification of novel gene functions. Metabolomics and transcriptomics data of Arabidopsis grown under sulfur deficiency were combined and analyzed by batch-learning self-organizing mapping. A group of metabolites/genes regulated by the same mechanism clustered together. The metabolism of glucosinolates was shown to be coordinately regulated. Three uncharacterized putative sulfotransferase genes clustering together with known glucosinolate biosynthesis genes were candidates for involvement in biosynthesis. In vitro enzymatic assays of the recombinant gene products confirmed their functions as desulfoglucosinolate sulfotransferases. Several genes involved in sulfur assimilation clustered with O-acetylserine, which is considered a positive regulator of these genes. The genes involved in anthocyanin biosynthesis clustered with the gene encoding a transcriptional factor that up-regulates specifically anthocyanin biosynthesis genes. These results suggested that regulatory metabolites and transcriptional factor genes can be identified by this approach, based on the assumption that they cluster with the downstream genes they regulate. This strategy is applicable not only to plant but also to other organisms for functional elucidation of unknown genes.

Arabidopsis↗

PubMed1996

The gene cluster directing O-antigen biosynthesis in Yersinia enterocolitica serotype 0:8: identification of the genes for mannose and galactose biosynthesis and the gene for the O-antigen polymerase.

The rfb gene cluster of Yersinia enterocolitica serotype O:8 (YeO8) strain 8081-c was cloned by cosmid cloning. Restriction mapping, deletion analysis and transposon mutagenesis showed that about 19 kb of the cloned DNA is essential for the synthesis and expression of the YeO8 O-side-chain in Escherichia coli. Deletion analysis generated a derivative that expressed semi-rough LPS, a phenotype typical of an rfc mutant lacking the O-antigen polymerase. The deletions and transcomplementation experiments allowed localization of the rfc gene to the 3'-end of the rfb gene cluster. The deduced YeO8 Rfc did not share significant amino acid sequence similarity with any other protein, but its amino acid composition and hydrophobicity profile are similar to those of identified Rfc proteins. In addition, the codon usage of the rfc gene is similar to other rfc genes. Nucleotide sequence analysis identified three other genes upstream of rfc. Two of the gene products showed 60-70% identity to the RfbM and RfbK proteins that are biosynthetic enzymes for the GDPmannose pathway of enterobacteria. The third gene product was about 50-80% identical to the bacterial GalE protein, UDPglucose 4-epimerase, which catalyses the epimerization of UDPglucose to UDPgalactose. Since mannose and galactose are both present in the YeO8 O-antigen repeat unit, the above three genes are likely to belong to the rfb gene cluster. A gene similar to the gsk gene downstream of rfc, and genes similar to adk and hemH upstream of the rfb gene cluster, were recognized. Thus the rfb gene cluster of YeO8 is located between the adk-hemH and gsk loci, and the order is adk-hemH-rfb-rfc-gsk in the chromosome. Also in other Yersinia spp., the locus downstream of the hemH gene is occupied by gene clusters associated with LPS biosynthesis.

Amino Acid Sequence↗

PubMed2005

DNA methylation of genes linked with retinoid signaling in gastric carcinoma: expression of the retinoid acid receptor beta, cellular retinol-binding protein 1, and tazarotene-induced gene 1 genes is associated with DNA methylation.

BACKGROUND: Hypermethylation of CpG islands has been associated with silencing of various tumor suppressor genes, and the retinoid acid receptor beta (RARbeta), cellular retinol-binding protein 1 (CRBP1), and tazarotene-induced gene 1 (TIG1) genes have been associated with retinoic acid signaling. To the authors' knowledge, little is known regarding the involvement of these three genes in gastric carcinoma (GC). In this study, the authors investigated the methylation status of these genes and analyzed the role of their DNA methylation in GC. METHODS: DNA methylation of 3 retinoic acid-associated genes was analyzed in 42 samples of GC from 42 patients and in 8 GC cell lines by methylation-specific polymerase chain reaction (PCR) analysis. The mRNA expression levels for these three genes were measured by quantitative reverse transcription-PCR. RESULTS: In 7 of 8 GC cell lines, the CRBP1 gene was hypermethylated, and CRBP1 transcription was inactive. In 6 of 8 GC cell lines, the TIG1 gene was hypermethylated, and TIG1 transcription was inactive. Treatment with demethylating agent 5-aza-2'-deoxycytidine restored both CRBP1 and TIG1 transcription. DNA methylation of the RARbeta, CRBP1, and TIG1 genes was detected in 15 of 42 GC samples (36%), 14 of 42 GC samples (33%), and 4 of 42 GC samples (10%), respectively, and in 6 of 30 samples (20%), 0 of 30 samples (0%), and 1 of 30 samples (3%) of corresponding nonneoplastic mucosa. None of the 10 normal gastric mucosa samples from young, healthy individuals demonstrated hypermethylation of any of these genes. DNA methylation of each gene was associated significantly with low mRNA expression of the respective gene. Twenty-four of 42 GC samples (57%) demonstrated hypermethylation of at least 1 of the 3 genes. However, no significant, concordant hypermethylation of these genes was observed. CONCLUSIONS: The results suggested that gastric carcinogenesis involves transcriptional inactivation by aberrant DNA methylation of genes related to retinoid signaling.

Adenocarcinoma↗

PubMed1996

hrp gene-dependent induction of hin1: a plant gene activated rapidly by both harpins and the avrPto gene-mediated signal.

Two classes of bacterial genes are involved in the elicitation of the plant hypersensitive response (HR) in resistant plants: hrp genes and avr genes. hrp genes have been shown to be involved in the production and secretion of a new class of bacterial virulence/avirulence proteins, including harpin of Erwinia amylovora and harpinPss of Pseudomonas syringae. The ability of avr genes in the elicitation of the HR/resistance is dependent on functional hrp genes. The relationships between harpins and avr gene products are not known. This study investigates the plant genes induced by harpins and the effect of avr genes on the expression of such plant genes. A tobacco gene highly induced by harpins was isolated by a subtractive hybridization method. Induction of hin1 by P.s. pv. syringae 61 (Pss61) was found to be dependent on functional bacterial hrp genes. P. fluorescens (a saprophyte) or hrp mutants defective in the Hrp secretion pathway did not induce hin1 significantly. A hin 1-related gene in tomato cv. Rio Grande-PtoR was found to be rapidly induced by P. s. pv. tomato T1 (a virulent bacterium on Rio Grande-PtoR) containing the avrPto gene, which mediates the elictation of the HR/resistance in a Pto plant resistance gene-dependent manner. The induction of hin1 by bacteria correlates with production of harpins in planta. The putative open reading frame of hin1 encodes a novel protein of 221 amino acids. The data suggest that harpins and the avrPto-mediated signal induce a common plant gene in the elicitation of the HR.

Amino Acid Sequence↗

PubMed2006

Robust and comprehensive analysis of 20 osteoporosis candidate genes by very high-density single-nucleotide polymorphism screen among 405 white nuclear families identified significant association and gene-gene interaction.

UNLABELLED: Many "novel" osteoporosis candidate genes have been proposed in recent years. To advance our knowledge of their roles in osteoporosis, we screened 20 such genes using a set of high-density SNPs in a large family-based study. Our efforts led to the prioritization of those osteoporosis genes and the detection of gene-gene interactions. INTRODUCTION: We performed large-scale family-based association analyses of 20 novel osteoporosis candidate genes using 277 single nucleotide polymorphisms (SNPs) for the quantitative trait BMD variation and the qualitative trait osteoporosis (OP) at three clinically important skeletal sites: spine, hip, and ultradistal radius (UD). MATERIALS AND METHODS: One thousand eight hundred seventy-three subjects from 405 white nuclear families were genotyped and analyzed with an average density of one SNP per 4 kb across the 20 genes. We conducted association analyses by SNP- and haplotype-based family-based association test (FBAT) and performed gene-gene interaction analyses using multianalytic approaches such as multifactor-dimensionality reduction (MDR) and conditional logistic regression. RESULTS AND CONCLUSIONS: We detected four genes (DBP, LRP5, CYP17, and RANK) that showed highly suggestive associations (10,000-permutation derived empirical global p < or = 0.01) with spine BMD/OP; four genes (CYP19, RANK, RANKL, and CYP17) highly suggestive for hip BMD/OP; and four genes (CYP19, BMP2, RANK, and TNFR2) highly suggestive for UD BMD/OP. The associations between BMP2 with UD BMD and those between RANK with OP at the spine, hip, and UD also met the experiment-wide stringent criterion (empirical global p < or = 0.0007). Sex-stratified analyses further showed that some of the significant associations in the total sample were driven by either male or female subjects. In addition, we identified and validated a two-locus gene-gene interaction model involving GCR and ESR2, for which prior biological evidence exists. Our results suggested the prioritization of osteoporosis candidate genes from among the many proposed in recent years and revealed the significant gene-gene interaction effects influencing osteoporosis risk.

Adult↗

PubMed1987

Eleven MRL-lpr/lpr anti-DNA autoantibodies are encoded by genes from four VH gene families: a potentially biased usage of VH genes.

The genes encoding 11 independently derived anti-DNA autoantibodies from the lupus-prone mouse strain, MRL-lpr/lpr, were examined with VH, D, and JH gene probes. These autoantibodies do not define new VH gene families, since all of the autoantibodies were encoded by VH genes from four of the nine known gene families. A minimum of nine different VH genes encoded this panel of 11 anti-DNA autoantibodies. These results are consistent with the stochastic use of the VH gene repertoire and the expression of multiple VH genes. However, the data is also consistent with a biased usage of the VH gene repertoire. First, two pairs of autoantibodies, one from the J558 family and one from the 7183 family, appear to express identical or closely related VH genes as determined by the position of two restriction enzyme sites 5' of the expressed VH genes. In addition, three autoantibodies that appear to be sister clones might define a third VH gene that is used repeatedly. Secondly, about 45% of the panel is encoded by the Q52 and 7183 families, which are the 3' most families. These families have been shown to be preferentially rearranged early in B cell ontogeny. This suggests that some anti-DNA autoantibodies might originate from a population of B cells that predominate early in ontogeny. An alternative hypothesis is that the potential bias in VH gene and gene family usage could be due to antigen selection. All four JH genes are expressed, although the JH1 gene appears to be underutilized in both expressed and unexpressed rearrangements. Two members of the panel that bind double-stranded DNA were encoded by two different VH gene families, the S107 family and the J558 family.

Amino Acid Sequence↗

PubMed1994

Structure and genetics of the partially duplicated gene RP located immediately upstream of the complement C4A and the C4B genes in the HLA class III region. Molecular cloning, exon-intron structure, composite retroposon, and breakpoint of gene duplication.

The correlation of many HLA-associated autoimmune and genetic diseases with the polymorphic complement C4 genes may be attributed to the presence of disease susceptibility genes in the close proximity of C4. We have cloned and characterized a pair of partially duplicated genes, RP1 and RP2, located 611 base pairs upstream of the human C4A and C4B genes, respectively. The putative RP protein, consisting of 364 amino acid residues, is basic and highly hydrophilic. There is a bipartite nuclear localization signal at residues 114-131 and therefore RP may be a nuclear protein. Northern blot analysis suggested that RP is ubiquitously expressed. The 5' region of the RP1 gene is CpG rich, which is a characteristic of housekeeping genes. The RP1 gene contains nine exons. Located in the fourth intron is a cluster of Alu elements, and a newly defined composite retroposon SVA with a SINE, multiple copies of GC-rich VNTRs and an Alu element altogether enclosed by direct terminal repeats. Members of SVA are also present in the complement C2 gene located about 20 kilobases upstream of RP1 in the HLA and in the cytochrome CYP1A1 gene. Determination of the DNA sequences for RP2 from two different HLA haplotypes revealed identical hybrid sequences which resulted from fusion of RP with the tenascin-like Gene X and truncation of the 5' regions of both genes. Cumulative data suggest that the four tandemly arranged genes RP, complement C4, steroid 21-hydroxylase (CYP21), and Gene X altogether form a modular structure, RCCX. The number of RCCX modules varies from one to three or more in the population. Absence of the truncated genes RP2 and Gene XA have been detected in genomes with single RCCX modules. Duplication of the RCCX modules probably occurred before the speciation of great apes and humans as they contain the same breakpoint region of RP and Gene X gene duplication.

Alleles↗

PubMed2001

The mouse lens fiber-cell intrinsic membrane protein MP19 gene (Lim2) and granule membrane protein GMP-17 gene (Nkg7): Isolation and sequence analysis of two neighboring genes.

PURPOSE: The lens fiber cell intrinsic membrane protein MP19 appears to play a key role in lens fiber cell structure or communication, and thus cataractogenesis. The goal of this study was to isolate and characterize the entire gene structure of the MP19 gene, termed Lim2, and to investigate gene sequences surrounding this lens-specific gene. METHODS: A 129/SvJ mouse genomic DNA library was screened using radioisotope labeled bovine MP19 cDNA. From this screening, an 11 kb genomic fragment was isolated which contained the entire Lim2 gene, and a neighboring gene, Nkg7, which codes for a 17 kDa granulocyte membrane protein termed GMP-17. The nucleotide sequence of this entire fragment was obtained using double strand automated sequencing techniques. Using CAT and green fluorescent protein reporter constructs, Lim2 5'-upstream promoter sequences were analyzed. RESULTS: An 11,182 base pair genomic clone containing the entire murine Lim2 gene and another downstream gene, Nkg7, was obtained and completely sequenced. These two genes are only 1,182 base pairs apart, from the poly(A) signal of the Lim2 gene to the published transcriptional start site of Nkg7. Interestingly, the protein coded for by Nkg7, GMP-17, is very similar to the product of the lens Lim2 gene, MP19, in many respects. Both proteins are transmembrane proteins, with each having 4 transmembrane loops. The amino acid sequence of the two proteins is 34% identical, and 49% with respect to similar amino acids. The size of mouse Lim2 is 5,896 base pairs from the transcriptional start site to the poly(A) signal, and contains five exons and four introns. Exons 2-5 of the Lim2 gene encode a polypeptide of 173 amino acids, having over 92% identity to human MP19. Using chloramphenicol acetyltransferase (CAT) and green fluorescent protein (GFP) reporter constructs, it was determined that about 160 bp of sequence upstream from the start of transcription is both necessary and sufficient for efficient expression levels as well as tissue specificity of expression. CONCLUSIONS: The mouse Lim2 gene is very similar to the human LIM2 gene, both having the same number of exons and introns. The coding nucleotide sequences from both species are 88% identical, and 92% identical at the amino acid level. In the immediate 5'-upstream region of these two genes, several highly conserved regions are observed. Due to the similarity of the MP19 and GMP-17 proteins, it is interesting to speculate that the lens MP19 and the lymphocyte-associated GMP-17 may have originated from one primordial gene which, through genetic drift, resulted in two separate proteins having similar functions in two widely separated tissue types.

Amino Acid Sequence↗

PubMed1993

Sequence of a 7.8 kb segment on the left arm of yeast chromosome XI reveals four open reading frames, including the CAP1 gene, an intron-containing gene and a gene encoding a homolog to the mammalian UOG-1 gene.

We report here the DNA sequence of a segment of chromosome XI of Saccharomyces cerevisiae extending over 7.8 kb. The segment contains four long open reading frames, YKL150, YKL153, YKL155 and YKL156, YKL155 corresponds to the CAP1 gene. YKL153 contains an intron and shows an extremely biased codon usage suggestive of a highly expressed protein. YKL156 is a homolog to UOG-1, an open reading frame associated with the cDNA clone of the mammalian growth/differentiation factor 1. YKL150 reveals common motifs to both the RNA polymerase II elongation factor of Drosophila melanogaster and to the yeast PPR2 gene product.

Actin Capping Proteins↗

PubMed2001

Mitochondrial gene transfer in pieces: fission of the ribosomal protein gene rpl2 and partial or complete gene transfer to the nucleus.

Mitochondrial genes are usually conserved in size in angiosperms. A notable exception is the rpl2 gene, which is considerably shorter in the eudicot Arabidopsis than in the monocot rice. Here, we show that a severely truncated mitochondrial rpl2 gene (termed 5' rpl2) was created by the formation of a premature stop codon early in eudicot evolution. This 5' rpl2 gene was subsequently lost many times from the mitochondrial DNAs of 179 core eudicots surveyed by Southern hybridization. The sequence corresponding to the 3' end of rice rpl2 (termed 3' rpl2) has been lost much more pervasively among the mitochondrial DNAs of core eudicots than has 5' rpl2. Furthermore, where still present in these mitochondrial genomes, 3' rpl2 always appears to be a pseudogene, and there is no evidence that 3' rpl2 was ever a functional mitochondrial gene. An intact and expressed 3' rpl2 gene was discovered in the nucleus of five diverse eudicots (tomato, cotton, Arabidopsis, soybean, and Medicago). In the first three of these species, 5' rpl2 is still present in the mitochondrion, unlike the two legumes, where both parts of rpl2 are present in the nucleus as separate genes. The full-length rpl2 gene has been transferred intact to the nucleus in maize. We propose that the 3' end of rpl2 was functionally transferred to the nucleus early in eudicot evolution, and that this event then permitted the nonsense mutation that gave rise to the mitochondrial 5' rpl2 gene. Once 5' rpl2 was established as a stand-alone mitochondrial gene, it was then lost, and was probably transferred to the nucleus many times. This complex history of gene fission and gene transfer has created four distinct types of rpl2 structures or compartmentalizations in angiosperms: (1) intact rpl2 gene in the mitochondrion, (2) intact gene in the nucleus, (3) split gene, 5' in the mitochondrion and 3' in the nucleus, and (4) split gene, both parts in the nucleus.

Amino Acid Sequence↗

PubMed2006

[Synthesis of new gene-loaded microbubbles serve as gene delivery vehicle applied in reporter gene transfer into cardiac myocytes].

To improve the stability and gene-carried capability of gene-attached microbubbles, the method for manufacture of albumin microbubbles was modified and new gene-loaded microbubbles were synthesized by incorporated gene-PEI complex into the shell of microbubbles. Agarose gel electrophoresis and bacteria transformation showed that PEI had the ability to provide the protection of plasmid DNA from ultrasonic degradation. The new gene-loaded microbubbles exhibited excellent acoustical and hemorheological properties. Moreover, they could carry more plasmid DNA than gene-attached microbubbles. beta-galactosidase plasmid transfection into cardiac myocytes was performed by using ultrasound targeted destruction of new gene-loaded microbubbles or gene-attached microbubbles. Gene expression in cardiac myocytes was detected by beta-galactosidase in situ staining and quantitive assay. It was shown that beta-galactosidase activity in cardiac myocytes was enhanced 107-fold by ultrasonic destruction of gene-loaded microbubbles compared with naked plasmid transfection and new gene-loaded microbubbles resulted in 6.85-fold increase in beta-galactosidase activity compared with optimal transfection mediated by gene-attached microbubbles. These results suggested that ultrasonic destruction of the gene-loaded microbubbles can enhance the cardiac myocytes exogenous gene transfer efficiency significantly and new gene-loaded microbubbles is an efficient and safe gene delivery vehicle.

Animals↗