Biomedical subjects
Publications and source records attributed to D E Rancourt.
The Rancourt EST Database (RED) is a web-based system for the analysis, management, and dissemination of expressed sequence tags (ESTs). RED represents a flexible template DNA sequence database that can be easily manipulated to suit the needs of other laboratories undertaking mid-size sequencing projects.
A rate-determining step in gene targeting is the generation of the targeting vector. We have developed bacteriophage gene targeting vectorology, which shortens the timeline of targeting vector construction. Using retro-recombination screening, we can rapidly isolate targeting vectors from an embryonic stem cell genomic library via integrative and excisive recombination. We have demonstrated that recombination can be used to introduce specific point mutations or unique restriction sites into gene targeting vectors via transplacement. Using the choline/ethanolamine kinase alpha and beta genes as models, we demonstrate that transplacement can also be used to introduce specifically a neo resistance cassette into a gene targeting phage. In our experience, the lambdaTK gene targeting system offers considerable flexibility and efficiency in TV construction, which makes generating multiple vectors in one week's time possible.
Before implantation the blastocyst is maintained within a proteinaceous coat, the zona pellucida, which prevents polyspermy and ectopic pregnancy. An extracellular trypsin-like activity, which is necessary for hatching from the zona pellucida in vitro, is localized to the abembryonic pole of the blastocyst. Upon hatching, the extracellular matrix-degrading proteinases urokinase plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9) are thought to promote blastocyst invasion. However, gene disruption experiments have demonstrated that uPA and MMP-9 are dispensable and, thus, that other key enzymes are involved in implantation. In this study, a novel implantation serine proteinase (ISP1) gene, which is distantly related to haematopoietic tryptases and represents a novel branch of the S1 proteinase family, was cloned. ISP1 is expressed throughout morulae and blastocysts during hatching and outgrowth. Abrogation of ISP1 mRNA accumulation using antisense oligodeoxynucleotides disrupts blastocyst hatching and outgrowth in vitro. The results of this study indicate that the ISP1 gene probably encodes the long sought after 'hatching enzyme' that is localized to the abembryonic pole during hatching in vitro. ISP1 is the earliest embryo-specific proteinase to be expressed in implantation and may play a critical role in connecting embryo hatching to the establishment of implantation competence at the abembryonic pole of the blastocyst.
Hormones prepare the uterus for the arrival and subsequent invasion of the embryo during pregnancy. Extracellular matrix-degrading proteinases and their inhibitors are involved in this integration process. Recent genetic evidence indicates that there is redundancy within the implantation proteinase cascade, indicating that additional proteinases may be involved. Recently, we described a novel implantation serine proteinase (ISP1) gene that encodes the embryo-derived enzyme strypsin, which is necessary for blastocyst hatching in vitro and the initiation of invasion. The evidence presented in the present study indicates that a second proteinase secreted from the uterus also participates in lysis of the zona pellucida. A second implantation serine proteinase gene (ISP2) was isolated, which encodes a related secreted tryptase expressed specifically within uterine endometrial glands. In pseudopregnancy, ISP2 gene expression is dependent on progesterone priming and is inhibited by the antiprogestin RU486. On the basis of similarities between ISP2 gene expression and that of a progesterone-regulated luminal proteinase associated with lysis of the zona pellucida, it is possible that the strypsin-related protein, ISP2, may encode a zona lysin proteinase.
The mutant L-type calcium channel alpha(1)-subunit gene, CACNA1F, was recently identified as the gene responsible for incomplete X-linked congenital stationary night blindness. The 6070-bp mRNA transcript is predicted to encode a 1977-amino-acid pore-forming protein with cytoplasmic amino- and carboxyl-termini separated by four homologous repeat domains, each consisting of six transmembrane segments. CACNA1F has been shown to be preferentially expressed in the retina, indicative of a specific functional role in visual processing. We have established the complete sequence of the murine orthologue of CACNA1F, namely Cacna1f. The total length of the mRNA transcript of the murine gene was established to be 6080 bp with an open reading frame that translates into a 1985-amino-acid protein. Cacna1f is highly homologous to the human sequence, with 90% identity at the amino acid level and almost perfect conservation between the functional domains. Furthermore, as in the human gene, the 3' end of the Cacna1f gene maps within 5 kb of the 5' end of the mouse synaptophysin gene in a region orthologous to Xp11.23. Using in situ hybridization, Cacna1f was found to be expressed in the inner and outer nuclear layers and the ganglion cell layer of the retina.
Targeted gene disruption is an important tool in molecular medicine, allowing for the generation of animal models of human disease. Conventional methods of targeting vector (TV) construction are difficult and represent a rate limiting step in any targeting experiment. We previously demonstrated that bacteriophage are capable of acting as TVs directly, obviating the requirement for 'rolling out' plasmids from primary phage clones and thus eliminating an additional, time consuming step. We have also developed methods which facilitate the construction of TVs using recombination. In this approach, modification cassettes and point mutations are shuttled to specific sites in phage TVs using phage-plasmid recombination. Here, we report a further improvement in TV generation using a recombination screening-based approach deemed 'retro-recombination screening' (RRS). We demonstrate that phage vectors containing specific genomic clones can be genetically isolated from a lambdaTK embryonic stem cell genomic library using a cycle of integrative recombination and condensation. By introducing the gam gene of bacteriophage lambda into the probe plasmid it is possible to select for positive clones which have excised the plasmid, thus returning to their native conformation following purification from the library. Rapid clone isolation using the RRS protocol provides another method by which the time required for TV construction may be further reduced.
We have used a method for synchronously differentiating murine embryonic stem (ES) cells into functional neurons and glia in culture. Using subtractive hybridization we isolated approximately 1200 cDNA clones from ES cell cultures at the neural precursor stage of neural differentiation. Pilot studies indicated that this library is a good source of novel neuro-embryonic cDNA clones. We therefore screened the entire library by single-pass sequencing. Characterization of 604 non-redundant cDNA clones by BLAST revealed 96 novel expressed sequence tags (ESTs) and an additional 197 matching uncharacterized ESTs or genomic clones derived from genome sequencing projects. With the exception of a handful of genes, whose functions are still unclear, most of the 311 known genes identified in this screen are expressed in embryonic development and/or the nervous system. At least 80 of these genes are implicated in disorders of differentiation, neural development and/or neural function. This study provides an initial snapshot of gene expression during early neural differentiation of ES cell cultures. Given the recent identification of human ES cells, further characterization of these novel and uncharacterized ESTs has the potential to identify genes that may be important in nervous system development, physiology and disease.
Neurodegenerative diseases cost the economies of the developed world billions of dollars per annum. Given ageing population profiles and the increasing extent of this problem, there has been a surge of interest in neural stem cells and in neural differentiation protocols that yield neural cells for therapeutic transplantation. Due to the oncogenic potential of stem cells a better characterisation of neural differentiation, including the identification of new neurotrophic factors, is required. Stem cell cultures undergoing synchronous in vitro neural differentiation provide a valuable resource for gene discovery. Novel tools such as microarrays promise to yield information regarding gene expression in stem cells. With the completion of the yeast, C. elegans, Drosophila, human, and mouse genome projects, the functional characterisation of genes using genetic and bioinformatic tools will aid in the identification of important regulators of neural differentiation.
Site-specific mutagenesis provides the ability to alter DNA with precision so that the function of any given gene can be more fully understood. Several methods of in vitro mutagenesis are time-consuming and imprecise, requiring the subcloning and sequencing of products. Here we describe a rapid, high fidelity method of in situ mutagenesis in bacteriophage lambda using transplacement. Using this method, mutations are transferred from oligonucleotides to target phages using a plasmid interface. A small (50 bp) homology region bearing a centred point mutation is generated from oligonucleotides and subcloned into a transplacement plasmid bearing positive and negative phage selectable markers. Following a positive/negative selection cycle of integrative recombination and excision, the point mutation is transferred precisely from plasmid to phage in a subset ( approximately 25-50%) of recombinants. As the fidelity of both oligonucleotide synthesis and phage-plasmid recombination is great, this approach is extremely reliable. Using transplacement, point mutations can be accurately deposited within large phage clones and we demonstrate the utility of this technique in the construction of gene targeting vectors in bacteriophages.
The tumour suppressor p53 becomes activated as a transcription factor in response to DNA damage, but the mechanism for this activation is unclear. A good candidate for an upstream activator of p53 is the DNA-dependent protein kinase (DNA-PK) that depends on the presence of DNA breaks for its activity. Here we investigate the link between DNA damage and the activation of DNA-PK and of p53. To determine whether DNA-PK is an upstream mediator of the p53 DNA-damage response, we analysed a severe combined-immunodeficiency (SCID) mouse cell line, SCGR11, and the human glioma cell line M059J . Both cell lines lack any detectable DNA-PK activity. We find that p53 is incapable of binding to DNA in the absence of DNA-PK, that DNA-PK is necessary but not sufficient for activation of p53 sequence-specific DNA binding, and that this activation occurs in response to DNA damage. Our results establish DNA-PK as a link between DNA damage and p53 activation, and reveal the existence of a mammalian DNA-damage-response pathway.
Targeted mutagenesis is an extremely useful experimental approach in molecular medicine, allowing the generation of specialized animals that are mutant for any gene of interest. Currently the rate determining step in any gene targeting experiment is construction of the targeting vector (TV). In order to streamline gene targeting methods and avoid problems encountered with plasmid TVs, we describe the direct application of lambda phage in targeted mutagenesis. The recombination-proficient phage vector lambda2TK permits generation of TVs by conventional restriction-ligation or recombination-mediated methods. The resulting lambdaTV DNA can then be cleaved with restriction endonucleases to release the bacteriophage arms and can subsequently be electroporated directly into ES cells to yield gene targets. We demonstrate that in vivo phage-plasmid recombination can be used to introduce neo and lacZ - neo mutations into precise positions within a lambda2TK subclone via double crossover recombination. We describe two methods for eliminating single crossover recombinants, spi selection and size restriction, both of which result in phage TVs bearing double crossover insertions. Thus TVs can be easily and quickly generated in bacteriophage without plasmid subcloning and with little genomic sequence or restriction site information.
During mammalian embryogenesis, proteinases are important for both matrix remodeling and the activation of latent growth factors. As subtilisin-like prohormone convertases (SPCs) have recently been found to activate members of the matrix metalloproteinase and transforming growth factor-beta (TGF-beta) families, we sought to investigate the role of this gene family in murine implantation and embryogenesis. Using active site polymerase chain reaction (PCR) cloning, four members of the SPC family were identified at embryonic day 6.5: SPC1, SPC2, SPC3, and SPC6. In situ hybridization analysis of sectioned E6.5 embryos in utero demonstrated strong SPC6 expression in differentiated decidua, overlapping and extending beyond the region previously described for the metalloproteinase inhibitor TIMP-2. Lower levels of SPC6 expression were observed in trophoblasts and in the ectoplacental cone, suggesting multiple roles for this enzyme in implantation. Northern analysis showed that SPC6 mRNA in embryos is represented by two distinct sizes of message--the isoform SPC6-1 (3.0 kb) is most abundant at all stages, but significant levels of SPC6-b (6.0 kb) occur in E12.5 embryos. Whole mount in situ hybridization to E8.5 embryos demonstrated strong SPC6 expression in the most posterior somite. This somitic staining moved caudally with the developing embryo and by E10.5 became localized to the posterior of the tail, indicating that SPC6 is involved in somitogenesis. SPC6 was expressed at low levels throughout the embryo, except in the developing nervous system, and strong expression was observed in the first branchial arch and in skeletal regions of the developing vertebrae, limbs, and craniofacium, suggesting additional roles for SPC6 in skeletogenesis.
The synovial fluid or group II secretory phospholipase A2 (sPLA2) has been implicated as an important agent involved in a number of inflammatory processes. In an attempt to determine the role of sPLA2 in inflammation, we set out to generate sPLA2-deficient mice. During this investigation, we observed that in a number of inbred mouse strains, the sPLA2 gene was already disrupted by a frameshift mutation in exon 3. This mutation, a T insertion at position 166 from the ATG of the cDNA, terminates out of frame in exon 4, resulting in the disruption of the calcium binding domain in exon 3 and loss of both activity domains coded by exons 4 and 5. The mouse strains C57BL/6, 129/Sv, and B10.RIII were found to be homozygous for the defective sPLA2 gene, whereas outbred CD-1:SW mice had variable genotype at this locus. BALB/c, C3H/HE, DBA/1, DBA/2, NZB/BIN, and MRL lpr/lpr mice had a normal sPLA2 genotype. The sPLA2 mRNA was expressed at very high levels in the BALB/c mouse small intestine, whereas in the small intestine of the sPLA2 mutant mouse strains, sPLA2 mRNA was undetectable. In addition, PLA2 activity in acid extracts of the small intestine were approximately 40 times higher in BALB/c than in the mutant mice. Transcription of the mutant sPLA2 gene resulted in multiple transcripts due to exon skipping. None of the resulting mutant mRNAs encoded an active product. The identification of this mutation should not only help define the physiological role of sPLA2 but also has important implications in mouse inflammatory models developed by targeted mutagenesis.
hoxb-5 and hoxb-6 are adjacent genes in the mouse HoxB locus and are members of the homeotic transcription factor complex that governs establishment of the mammalian body plan. To determine the roles of these genes during development, we generated mice with a targeted disruption in each gene. Three phenotypes affecting brachiocervicothoracic structures were found in the mutant mice. First, hoxb-5- homozygotes have a rostral shift of the shoulder girdle, analogous to what is seen in the human Sprengel anomaly. This suggests a role for hoxb-5 in specifying the position of limbs along the anteroposterior axis of the vertebrate body. Second, hoxb-6- homozygotes frequently have a missing first rib and a bifid second rib. The third phenotype, an anteriorizing homeotic transformation of the cervicothoracic vertebrae from C6 through T1, is common to both hoxb-5- and hoxb-6- homozygotes. Quite unexpectedly, hoxb-5, hoxb-6 transheterozygotes (hoxb-5-hoxb-6+/hoxb-5+ hoxb-6-) also show the third phenotype. By this classical genetic complementation test, these two mutations appear as alleles of the same gene. This phenomenon is termed nonallelic noncomplementation and suggests that these two genes function together to specify this region of the mammalian vertebral column.
Transgenic Drosophila melanogaster that contain a winter flounder antifreeze protein (AFP) gene fused to the transcriptional and translational control sequences of the host heat shock protein 70 gene express the transgene under heat shock conditions. They secrete into the hemolymph small quantities of a protein that reacts with antisera to AFP and is of a similar size to the proAFP precursor. To facilitate purification and characterization of this precursor, transformed fly lines homozygous for inserts on the 2nd, 3rd and X chromosomes were crossed together to generate a line with five and six AFP genes present in males and females, respectively. AFP production in the multi-gene line was approximately equal to the sum of that observed in the three starting lines and was just sufficient to perturb the growth of ice crystals. The AFP component was purified from heat-denatured hemolymph of this line by cation- and anion-exchange chromatography, followed by reverse-phase HPLC. Edman degradation sequencing of the purified protein showed that its N-terminus began two amino acids in from the predicted signal peptide cleavage point. An additional amino acid sequence was present that began two amino acids further into the 'pro' sequence. These AFP products are consistent with processing of the proAFP in Drosophila by a type IV dipeptidyl aminopeptidase, as has been suggested for processing in flounder.
The expression of fusion gene constructs containing Drosophila regulatory sequences and the structural portions of fish antifreeze protein genes have been examined by transfer into Drosophila melanogaster using P elements. A fusion gene, containing the enhancer, promoter, and cap site of the yolk polypeptide 1 gene, joined in the 5'-untranslated region to the structural portion of the winter flounder type I antifreeze gene, was transcribed in mature female transformants to give an mRNA of the predicted size, but no antifreeze protein was detected by Western blotting. When the same antifreeze protein gene was fused to a Drosophila hsp 70 gene regulatory region and placed downstream of the yolk polypeptide gene enhancer, appropriate expression of mRNA was directed by both gene regulatory elements. However, a translation product from this mRNA was only observed under heat shock conditions and was present at low levels. It is suggested that type I antifreeze mRNA, with its high content of alanine codons and their grouping into clusters of up to seven in a row, is poorly translated when in competition with other host mRNAs. In agreement with this hypothesis, a fusion gene construct between the yolk protein gene regulatory region and two type III antifreeze protein genes produced sub-mmolar concentrations of antifreeze protein in mature females from each of several transgenic lines analysed. The type III antifreeze protein does not have an imbalanced amino acid composition or sequence irregularities, and may be an appropriate choice for conferring freeze protection to frost-susceptible hosts by gene transfer.
We have expressed two antifreeze protein genes from the Atlantic wolffish, Anarhichas lupus, in Drosophila melanogaster by placing them under the divergent transcriptional control of the host yolk polypeptide (1 and 2) gene promoters. Both genes were joined to the central promoter region by fusion within the DNA encoding the signal polypeptides. The chimeric genes were introduced into flightless mutant Drosophila through P-element transformation. Transformed adult females from individual lines accumulated 1.5-5 mg/ml of antifreeze protein in their hemolymph. The protein was purified to homogeneity from hemolymph following thermal denaturation, step elution from SP-Sephadex, and reverse-phase HPLC. It was recovered in high yield and retained full biological activity even though one of the two gene fusions gave rise to a seven amino acid N-terminal extension on its antifreeze protein product.
The gene coding for the most abundant antifreeze protein (AFP) in the winter flounder was placed downstream of the Drosophila melanogaster hsp70 promoter and introduced into the D. melanogaster germ line by P-element-mediated transformation. In each of six transgenic strains tested, heat shock treatment induced the expression of two major AFP gene transcripts and one minor one. All three transcripts were spliced despite the lack of an obvious D. melanogaster internal intron-splicing sequence. The variation in transcript length was caused by selection of different polyadenylation sites. Western blots showed the presence of immunoreactive AFP in hemolymph from heat-shocked transformants. The immunoreactive material had a molecular weight of 6,200, which is consistent with the loss of the signal sequence from the primary translation product and the retention of the pro sequence. Thus, all the signals for flounder pre-mRNA and preprotein processing were recognized in D. melanogaster.