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PubMed2006

MgSlt2, a cellular integrity MAP kinase gene of the fungal wheat pathogen Mycosphaerella graminicola, is dispensable for penetration but essential for invasive growth.

Among expressed sequence tag libraries of Mycosphaerella graminicola isolate IPO323, we identified a full-length cDNA clone with high homology to the mitogen-activated protein (MAP) kinase Slt2 in Saccharomyces cerevisiae. This MAP kinase consists of a 1242-bp open reading frame, and encodes a 414-amino-acid protein. We designated this homolog MgSlt2, generated MgSlt2 knockout strains in M. graminicola isolate IPO323, and found several altered phenotypes in vitro as well as in planta. In yeast glucose broth, MgSlt2 disruptants showed a defective polarized growth in the tip cells upon aging, causing substantial local enlargements culminating in large swollen cells containing two to four nuclei. The MgSlt2 disruptants showed a significantly increased sensitivity to several fungicides, including miconazole (2x), bifonazole (>4x), imazalil (5x), and cyproconazole (10x), and were hypersensitive to glucanase. Unlike the wild type, MgSlt2 disruptants did not produce aerial mycelia and did not melanize on potato dextrose agar. Although cytological analysis in planta showed normal penetration of wheat stomata by the germ tubes of the MgSlt2 disruptants, subsequently formed hyphal filaments frequently were unable to branch out and establish invasive growth resulting in highly reduced virulence, and prevented pycnidia formation. Therefore, we conclude that MgSlt2 is a new pathogenicity factor in M. graminicola.

Amino Acid Sequence↗

PubMed2005

Novel ganglioside antigen identified by B cells in human medullary breast carcinomas: the proof of principle concerning the tumor-infiltrating B lymphocytes.

The potential tumor-recognizing capacity of B cells infiltrating human breast carcinoma is an important aspect of breast cancer biology. As an experimental system, we used human medullary breast carcinoma because of its heavy B lymphocytic infiltration paralleled to a relatively better prognosis. Ig-rearranged V region V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda genes, amplified by RT-PCR of the infiltrating B cells, were cloned, sequenced, and subjected to a comparative DNA analysis. A combinatorial single-chain variable fragment Ab minilibrary was constructed out of randomly selected V(H) and Vkappa clones and tested for binding activity. Our data analysis revealed that some of the V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda region sequences were being assigned to clusters with oligoclonal predominance, while other characteristics of the Ab repertoire were defined also. A tumor-restricted binder clone could be selected out of the single-chain variable fragment kappa minilibrary tested against membrane fractions of primary breast tumor cells and tumor cell lines, the V(H) of which proved to be the overexpressed V(H)3-1 cluster. The specific binding was confirmed by FACS analysis with primary breast carcinoma cells and MDA-MB 231 cell line. ELISA and thin layer chromatography dot-blot experiments showed this target Ag to be a ganglioside D3 (GD3). Our results are a proof of principle about the capacity of B cells infiltrating breast carcinomas to reveal key cancer-related Ags, such as the GD3. GD3-specific Abs may influence tumor cell progression and could be used for further development of diagnostic and/or therapeutic purposes.

Animals↗

PubMed2006

Characterization of gene expression profiles of normal canine retina and brain using a retinal cDNA microarray.

PURPOSE: Construction of a canine retinal custom cDNA microarray for comprehensive retinal gene expression profiling and application for the identification of genes that are preferentially expressed in the retina and brain lobes using a brain pool reference tissue. METHODS: A cDNA microarray was constructed utilizing clones obtained from a normalized canine retinal expressed sequence tag library. Gene expression profiles were analyzed for normal retina, as well as the cortex of the frontal, occipital, and temporal brain regions. Each sample was studied against a reference sample of pooled brain RNA. Data from a quantified scanned image were normalized using the loess subgrid procedure. Retina-enriched genes were identified using the Significance Analysis of Microarrays (SAM) algorithm, and confirmed by northern blot analyses for selected genes. Differences between biological samples were displayed using principal component analysis (PCA). RESULTS: Expression profiles for each tissue set were analyzed against the common reference of pooled brain. Changes in expression between the sample and the reference were higher in the retina (27.9%) than the individual brain tissues (2-6.6%). Furthermore, all individual retinal samples were clearly separated from any of the hybridizations using brain tissue in the PCA. The accuracy of observed changes in expression has been confirmed by northern blot analysis using five randomly chosen genes that represented a wide range of different expression levels between retina and brain. CONCLUSIONS: We have established an accurate and robust microarray system suitable for the investigation of expression patterns in the retina and brain. Characterization of the gene expression profiles in normal retina will facilitate the understanding of the processes that underline differences between normal and diseased retinas.

Animals↗

PubMed2003

[Construction of a SV40 promoter specific artificial transcription factor].

Transcriptions are regulated by transcription factors. Natural transcription factors usually consist of at least two functional domains: a DNA-binding domain and an effector domain. According to this, novel artificial transcription factors are designed to up or down regulate transcription and expression of a target gene. The Cys2-His2 zinc finger domain is a DNA-binding module that has been widely used as the DNA-binding domain in artificial transcription factors. Each zinc finger domain, which comprises about 30 amino acids that adopt a compact structure by chelating a zinc ion, typically functions by binding 3 base pairs of DNA sequence. Several zinc fingers linked together would bind proportionally longer DNA sequences. According to the "bipartite complementary" library strategy, a pair of zinc finger phage display libraries were constructed. After construction of the libraries, a 9bp sequence (5'-GCAGAGGCC-3') on the promoter of SV40 was chosen as a target for next step. After parallel selection, PCR amplification, desired fragments recovery, re-ligation, and additional rounds of selection, phage enzyme-linked ELISA experiments were performed to identify specific binding clones displaying the zinc fingers with predetermined sequence-specificity to our target sequence. Then two clones with strong ELISA signals were chosen to be tested for binding both to its full target site (5'-GCAGAGGCC-3') and to sites containing single transition mutations. The binding specificity of one of the two clones (clone 3) was shown to be fairly good. The three-finger DNA-binding domain targeted to SV40 promoter, that is, zinc finger sequences on clone 3, was fused to KOX1 suppression domain KRAB and cloned into pcDNA3.1 (+) (which expression product was artificial transcription factor). The zinc fingers (which expression product was the DNA-binding domain of artificial transcription factor) and KRAB domain only (which expression product was effector domain of artificial transcription factor) were also cloned separately into the same expression vector. All constructs contained an N-terminal nuclear localization signal. Every of the vectors (including pcDNA3.1 (+) without inserting sequences) were cotransfected with pGL3-Control and pRL-TK and the activity of luciferase was used to indicate the function of product from transfected expression vectors. Our artificial transcription factor was proved to repress the expression of reporter gene efficiently,while with only DNA-binding domain or effector domain the repression was not remarkable. By adding different effector domains and changing the DNA-binding domain, artificial transcription factor would have a wide range of potential applications.

Enzyme-Linked Immunosorbent Assay↗

PubMed1999

PEDB: the Prostate Expression Database.

The Prostate Expression Database (PEDB) is a curated relational database and suite of analysis tools designed for the study of prostate gene expression in normal and disease states. Expressed Sequence Tags (ESTs) and full-length cDNA sequences derived from more than 40 human prostate cDNA libraries are maintained and represent a wide spectrum of normal and pathological conditions. Detailed library information including tissue source, library construction methods, sequence diversity and abundance are available in a library archive. Prostate ESTs are assembled into distinct species groups using the multiple alignment program CAP2 and are annotated with information from the GenBank, dbEST and Unigene public sequence databases. Annotated sequences in PEDB are searched using the BLAST algorithm. The differential expression of each EST species can be viewed across all libraries using a Virtual Expression Analysis Tool (VEAT), a graphical user interface written in Java for intra- and inter-library species comparisons. PEDB may be accessed via the World Wide Web at http://www.mbt.washington.edu/PEDB/

Base Sequence↗

PubMed1987

An mRNA encoding poly-ubiquitin in porcine corpus luteum: identification by cDNA cloning and sequencing.

A cDNA library derived from a particular fraction of mRNA of porcine corpus luteum was screened by hybrid-release. cDNA clone pCL208 selected an mRNA which, in cell-free translation, directed the synthesis of a major polypeptide of 28 kD. Sequence analysis of clone pCL208 revealed that the entire coding sequence contained three complete repeats and a fourth incomplete repeat of porcine ubiquitin, joined head-to-tail without any spacer peptide sequences. The ultimate ubiquitin molecule contained a Phe residue as a carboxy-terminal extension. The coding sequence terminated with the stop codon TAA followed by a short untranslated region, the polyadenylation signal AATAAA, and a tract of A residues. Northern analysis indicated the presence of two types of ubiquitin-specific mRNAs, i.e., 1100 and 2500 bp, in porcine corpus luteum.

Amino Acid Sequence↗

PubMed2005

Insights into the genomes of archaea mediating the anaerobic oxidation of methane.

The anaerobic oxidation of methane is a globally significant process which is mediated by consortia of yet uncultivated methanotrophic archaea (ANME) and sulfate-reducing bacteria. In order to gain deeper insights into genome characteristics of the different ANME groups, large-insert genomic libraries were constructed using DNA extracted from a methanotrophic microbial mat growing in the anoxic part of the Black Sea, and from sediments above gas hydrates at the Hydrate Ridge off the coast of Oregon. Analysis of these fosmid libraries with respect to archaeal 16S rRNA gene diversity revealed a single ANME-1b ribotype for the Black Sea libraries, whereas the sequences derived from the Hydrate Ridge library phylogenetically affiliated with the ANME-2a, ANME-2c and ANME-3 group. Genome walking for ANME-1b resulted in a contiguous 155 kb composite genome fragment. The comparison of a set of four genomic fragments belonging to the different ANME groups revealed differences in the rRNA operon structure and the average G+C content, with the ANME-2c contig showing the highest divergence within the set. A detailed analysis of the ANME contigs with respect to genes putatively involved in the anaerobic oxidation of methane led to the identification of: (i) a putative N5,N10-methenyltetrahydromethanopterin cyclohydrolase gene, (ii) a gene cluster supposedly encoding a novel type of heterodisulfide reductase/dehydrogenase complex and (iii) a gene cluster putatively encoding a new type of CO dehydrogenase/acetyl-CoA synthase enzyme complex.

Amino Acid Sequence↗

PubMed1983

Retrieval of human DNA from rodent-human genomic libraries by a recombination process.

Human Alu repeat ("BLUR") sequences have been cloned into the mini-plasmid vector piVX. The resulting piBLUR clones have been used to rescue selectively, by recombination, bacteriophage carrying human DNA sequences from genomic libraries constructed using DNA from rodent-human somatic cell hybrids. piBLUR clones are able to retrieve human clones from such libraries because at least one Alu family repeat is present on most 15 to 20 kb fragments of human DNA and because of the relative species-specificity of the sequences comprising the Alu family. The rapid, selective plaque purification achieved results in the construction of a collection of recombinant phage carrying diverse human DNA inserts from a specific subset of the human karyotype. Subfragments of two recombinants rescued from a mouse-human somatic cell hybrid containing human chromosomes X, 10, 13, and 22 were mapped to human chromosomes X and 13, respectively, demonstrating the utility of this protocol for the isolation of human chromosome-specific DNA sequences from appropriate somatic cell hybrids.

Animals↗

PubMed1999

High throughput direct end sequencing of BAC clones.

Libraries constructed in bacterial artificial chromosome (BAC) vectors have become the choice for clone sets in high throughput genomic sequencing projects primarily because of their high stability. BAC libraries have been proposed as a source for minimally over-lapping clones for sequencing large genomic regions, and the use of BAC end sequences (i.e. sequences adjoining the insert sites) has been proposed as a primary means for selecting minimally overlapping clones for sequencing large genomic regions. For this strategy to be effective, high throughput methods for BAC end sequencing of all the clones in deep coverage BAC libraries needed to be developed. Here we describe a low cost, efficient, 96 well procedure for BAC end sequencing. These methods allow us to generate BAC end sequences from human and Arabidoposis libraries with an average read length of >450 bases and with a single pass sequencing average accuracy of >98%. Application of BAC end sequences in genomic sequen-cing is discussed.

Arabidopsis↗

PubMed1996

Identification of a group-I intron within the 25S rDNA from the yeast Arxula adeninivorans.

The 25S rDNA of the yeast Arxula adeninivorans LS3 has been closed from a genomic library and sequenced. This DNA could be localized on chromosome 1 from A. adeninivorans and comprised 3790 bp. The DNA sequence from this rDNA of the strain LS3 is very similar to the 25S rDNA of Candida albicans (91.7%), Saccharomyces cerevisiae (90.5%), Schizosaccharomyces pombe (83.8%) and Mucor racemosus (79.2%). Additionally a 411 bp insertion could be localized within the 25S rDNA. This intervening sequence, which is devoid of any long open reading frame, is a group-IC intron as revealed from its site of insertion, predicted secondary structure, and its self-splicing capability. The Arxula intron is intermediate in structure and sequence between the ribosomal introns of Tetrahymena thermophila and C. albicans.

Base Sequence↗

PubMed1998

Targeting of the domain-specific integral membrane protein PM52 to the periacrosomal plasma membrane during guinea pig spermiogenesis.

The sperm plasma membrane is segregated into functionally, biochemically, and structurally distinct domains yet the protein sorting pathways and assembly mechanisms that assemble these domains during spermiogenesis are incompletely understood. We previously characterized two structurally related size-variant, integral membrane proteins of 52 kDa (PM52) and 35 kDa localized to the periacrosomal plasma membrane of guinea pig cauda epididymal spermatozoa (Westbrook-Case et al., 1994). In this study we used light and electron microscopic immunocytochemistry to define the expression pattern and sorting pathway that establishes the domain-specific distribution of PM52 during spermiogenesis. The PM52 is first expressed in acrosome-phase spermatids and it localizes exclusively to the cytoplasmic lobe. Immunoelectron microscopy revealed that both cytoplasmic vesicles and the plasma membrane of the cytoplasmic lobe labeled with anti-PM52. During early stages of expression, PM52 appeared to be absent from the head region, but significant PM52 accumulation over the spermatid head was noted in late acrosomal phase spermatids. Throughout spermiogenesis PM52 extended posteriorly to the annulus, which represents a barrier preventing PM52 diffusion into the posterior tail. Following the migration of the annulus to the midpiece-principal piece junction, PM52 began to disappear from the flagellar region and at the completion of spermiogenesis most of the PM52 was restricted to the acrosomal segment. Spermatids and epididymal sperm PM52 exhibited identical sizes by SDS-PAGE and immunoblotting, indicating that they are not proteolytically modified during epididymal maturation. The PM52 antibodies were also used to screen a guinea pig testis cDNA library, and sequence determination of full-length PM52 clones demonstrated identity of a sperm membrane protein recently termed "sperad" (Quill and Garbers, 1996). Membrane barriers and potential mechanisms establishing the domain-specific residence of PM52 are discussed.

Acrosome↗

PubMed2001

Novel sperm-binding proteins of epididymal origin contain four fibronectin type II-modules.

Novel fibronectin type II (Fn2)-module proteins were cloned from human and canine epididymal cDNA libraries. cDNA sequences predicted a highly conserved protein family, related but not homologous to ungulate seminal plasma proteins (approximately 50% sequence identity), and the first known examples of proteins with four tandemly arranged Fn2-domains. By Northern blot and in situ hybridization analyses the encoding mRNAs were shown to be abundant products of the epididymal duct epithelium, but not detectable in other tissues. Homologous mRNAs were identified in the epididymides of various mammals, representing members of this novel protein family of epididymal origin. Within the Fn2-module-encoding stretches, species homologues displayed >85% sequence identity, but showed high variability at their predicted N-termini. An antipeptide antiserum in Western blot analyses detected 30-35 kDa immunoreactive protein bands in epididymal tissue, cauda epididymidal fluid, and sperm membrane protein preparations. The tandem arrangement of increasing numbers of Fn2-modules might functionally correspond to the tendency to form oligomers that has been described for lipid-binding proteins.

Amino Acid Sequence↗

PubMed1987

Repertoire of the human T cell gamma genes: high frequency of nonfunctional transcripts in thymus and mature T cells.

To further understand the structural diversity and function of the human T cell-specific gamma gene, 20 human gamma gene cDNA from both thymocyte and peripheral blood T lymphocyte libraries were sequenced and analyzed. Evidence of greater germ-line multiplicity and more extensive use of junctional flexibility, N-region diversity and perhaps D gamma gene segment incorporation was observed. In spite of the larger gamma gene repertoire found in humans compared to mice, the potential of the human gamma gene message to encode a functional product appears to be severely limited. In addition to the extremely low levels of gamma gene expression observed, each of the 20 gamma gene cDNA exhibit some form of deleterious mutation defect. This is in sharp contrast to the finding that human and mouse T cell antigen receptor alpha and beta messages isolated from various sources have functional coding potential in most cases. Thus, the role of the human T cell gamma gene becomes even more uncertain than before.

Amino Acid Sequence↗

PubMed1986

Molecular cloning of two new HPV types (HPV 37 and HPV 38) from a keratoacanthoma and a malignant melanoma.

Several benign and malignant skin tumors were analyzed for the presence of human papillomavirus (HPV) DNA. By hybridization with different HPV DNA probes under non-stringent conditions (Tm -40 degrees C), two tumors were found to contain HPV-specific DNA sequences in high copy numbers: (1) a keratoacanthoma from a patient who also suffered from a basalioma; (2) a superficial spreading malignant melanoma of an immunosuppressed patient. For further analysis of these DNA sequences genomic libraries from both tumor DNAs were constructed and, out of these, 4 different HPV DNA types have been cloned. By cross-hybridization experiments and restriction map analysis HPV 9 DNA was identified in the keratoacanthoma whereas HPV 17a DNA could be cloned from the malignant melanoma. From each tumor one additional HPV-type not identical to other known HPV-types was cloned. These isolates are closely related to HPV 9, 15, 17, 22 and 23. A physical map of both HPV DNAs was constructed. Size (7.8 kbp), co-linear alignment to HPV 16, cross-hybridization with other HPV-types under conditions of low stringency and monomeric episomal state of the HPV molecules indicate that these two DNA probes represent new HPV types that have been tentatively designated as HPV 37 (keratoacanthoma) and HPV 38 (malignant melanoma). None of these two HPV types could be found in any other of 231 tumor DNAs originating from different tissues.

Cloning, Molecular↗

PubMed1993

Thymosin beta-10 expression in melanoma cell lines and melanocytic lesions: a new progression marker for human cutaneous melanoma.

When screening a subtraction library for sequences that were specifically expressed in highly metastatic human melanoma cell lines, a cDNA clone was isolated encoding thymosin beta-10. We found that expression of thymosin beta-10 mRNA was associated with metastatic behavior of various human melanoma cell lines in nude mice. Furthermore, Northern blot analysis showed that also in freshly harvested human melanocytic lesions thymosin beta-10 was differentially expressed. Although expression of thymosin beta-10 was also examined in other non-melanoma model systems and materials, no clear relation could be established with metastatic potential or malignancy. Therefore, we conclude that thymosin beta-10 can be considered as a new progression marker for human cutaneous melanoma.

Amino Acid Sequence↗

PubMed1984

Evolution of a multigene family of V kappa germ line genes.

We have isolated a series of related V kappa germ line genes from a BALB/c sperm DNA library. DNA sequence analysis of four members of this V kappa 24 multigene family implies that three V kappa genes are functional whereas the fourth one (psi V kappa 24) is a pseudogene. The prototype gene (V kappa 24) encodes the variable region gene segment expressed in an immune response against phosphorylcholine. The other two functional genes (V kappa 24A and V kappa 24B) may be expressed against streptococcal group A carbohydrate. The time of divergence of the four genes was estimated by the rate of synonymous nucleotide changes. This implies that an ancestral gene has duplicated approximately 33-35 million years ago and a subsequent gene duplication event has occurred approximately 23 million years ago.

Amino Acid Sequence↗

PubMed2005

Characterization of five evolutionary conserved regions of the human tyrosine hydroxylase (TH) promoter: implications for the engineering of a human TH minimal promoter assembled in a self-inactivating lentiviral vector system.

A DNA fragment of about 13 kb containing the human tyrosine hydroxylase (TH) promoter was previously isolated from a genomic DNA library and sequenced. The 11 kb from the transcription start of the human TH promoter was successively joined to the green fluorescent protein (GFP) to generate a transgenic mouse model. High levels of GFP expression could be observed in TH-positive cells of the Substantia nigra of embryonic and adult mice. Intriguingly, the sequence of the human TH promoter showed a low degree of homology with the mouse and rat TH promoters. In fact, comparative analysis of the sequences of human, rat, and mouse TH promoters revealed only five small regions of high homology. These five evolutionarily conserved regions were numbered in numeric progression from the 5' end of human TH promoter. In the present study, a panel of minimal human TH promoters was generated to analyze the transcriptional activity and specificity of gene expression conferred by the five conserved regions (CRs). The series of constructs was termed 250 bp and contained the first -194 bp of the human TH promoter immediately upstream of the transcription start, the first 35 bp the human TH messenger RNA leader, plus one or more of the five CRs. All the constructs were assembled in a self-inactivating form of the latest series of lentiviral vector system based on the human immunodeficiency virus type 1 (HIV-1). Lentiviral-mediated gene transfer was highly efficient for the in vitro transduction of human neuronal progenitor cells (hNPCs). Since a subset of hNPCs express TH following in vitro treatment with a mixture of differentiating agents, it was possible to assess specificity of expression for all the minimal human TH promoters. Overall, the successive addition of the five conserved regions produced a greater degree of specificity in induced TH-positive hNPCs, in particular after the addition of CRI (-8,917, -8,876). However, the human TH minimal promoters did not show any specificity for TH-positive differentiated mouse primary striatal and S. nigra cells, indicating a difference of TH gene regulation between the human and mouse systems. The human TH minimal promoters may provide the opportunity for the selection of TH-positive human embryonic and adult stem cells for brain transplantation experiments in animal models for Parkinson's disease.

Animals↗

PubMed2005

Matrix metalloproteinase-2 (MMP-2) expression and regulation by tumor necrosis factor alpha (TNFalpha) in the bovine corpus luteum.

Angiogenesis and tissue remodeling events in the corpus luteum (CL) are mediated by matrix metalloproteinases (MMPs). We have recently reported the cloning of bovine membrane-type 1 metalloproteinase (MT1-MMP) and have shown that active MT1-MMP is correlated to MMP-2 activity in the CL during the estrous cycle. Given the important role that MMP-2 plays in neovascularization, we became interested in understanding the role of this enzyme in the CL, a system in which angiogenesis is exquisitely regulated in the course of its lifespan. The aims of the present study were to clone bovine MMP-2 cDNA, to investigate its temporal and spatial expression in three stages of CL during the estrous cycle and to study its regulation by TNFalpha, a key cytokine regulator of CL physiology. Bovine MMP-2 cDNA was isolated from a UNI-ZAP II bovine capillary endothelial cell cDNA library and sequenced. This gene encoded a protein of 662 amino acids. Luteal tissues were collected from non-lactating dairy cows on days 4, 10, and 16 of the estrous cycle. Northern and Western blotting revealed that the levels of MMP-2 mRNA (3.1 kb) and immunoreactive pro-MMP-2 protein (68 kDa) did not differ (P > 0.05) in any age of CL examined. In addition to large luteal cells, MMP2 was localized to endothelial cells in all ages of CL by immunohistochemistry. Studies using in vitro luteal cell cultures showed that MMP-2 mRNA, protein expression and activity was upregulated by TNFalpha in a dose- and time-dependent manner. The present study suggests that MMP-2 is predominantly produced by large luteal cells and endothelial cells, and that it plays an essential role in luteal remodeling and angiogenesis. These data also suggest that cytokines such as TNFalpha may modulate these processes by regulating MMP-2 expression.

Animals↗