Biomedical subjects

C S Shashikant

Publications and source records attributed to C S Shashikant.

At least 19 recordsLinked to original sources

PubMed2000

A recombinogenic targeting method to modify large-inserts for cis-regulatory analysis in transgenic mice: construction and expression of a 100-kb, zebrafish Hoxa-11b-lacZ reporter gene.

The identification of cis-sequences responsible for spatiotemporal patterns of gene expression often requires the functional analysis of large genomic regions. In this study a 100-kb zebrafish Hoxa-11b-lacZ reporter gene was constructed and expressed in transgenic mice. PAC clone 10-O19, containing a portion of the zebrafish HoxA-b cluster, was captured into the yeast-bacterial shuttle vector, pPAC-ResQ, by recombinogenic targeting. A lacZ reporter gene was then inserted in-frame into exon 1 of the zfHoxa-11b locus by a second round of recombinogenic targeting. Expression of the zfHoxa-11b-lacZ reporter gene in 10.5 d.p.f. transgenic mouse embryos was observed only in the posterior portion of the A-P axis, in the paraxial mesoderm, neural tube, and somites. These findings demonstrate the utility of recombinogenic targeting for the modification and expression of large inserts captured from P1/PAC clones.

Animals↗

PubMed1999

Mammalian Trithorax and polycomb-group homologues are antagonistic regulators of homeotic development.

Control of cell identity during development is specified in large part by the unique expression patterns of multiple homeobox-containing (Hox) genes in specific segments of an embryo. Trithorax and Polycomb-group (Trx-G and Pc-G) proteins in Drosophila maintain Hox expression or repression, respectively. Mixed lineage leukemia (MLL) is frequently involved in chromosomal translocations associated with acute leukemia and is the one established mammalian homologue of Trx. Bmi-1 was first identified as a collaborator in c-myc-induced murine lymphomagenesis and is homologous to the Drosophila Pc-G member Posterior sex combs. Here, we note the axial-skeletal transformations and altered Hox expression patterns of Mll-deficient and Bmi-1-deficient mice were normalized when both Mll and Bmi-1 were deleted, demonstrating their antagonistic role in determining segmental identity. Embryonic fibroblasts from Mll-deficient compared with Bmi-1-deficient mice demonstrate reciprocal regulation of Hox genes as well as an integrated Hoxc8-lacZ reporter construct. Reexpression of MLL was able to overcome repression, rescuing expression of Hoxc8-lacZ in Mll-deficient cells. Consistent with this, MLL and BMI-I display discrete subnuclear colocalization. Although Drosophila Pc-G and Trx-G members have been shown to maintain a previously established transcriptional pattern, we demonstrate that MLL can also dynamically regulate a target Hox gene.

Animals↗

PubMed1999

Direct cloning of genomic DNA by recombinogenic targeting method using a yeast-bacterial shuttle vector, pClasper.

We have developed a method to clone genomic DNA selectively into a yeast-bacterial shuttle vector, pClasper, by recombinogenic targeting in yeast. A gene-specific pClasper targeting vector was constructed with small recombinogenic ends (500 bp) derived from flanking sequences of the genomic region to be cloned. Linearized, recombinogenic pClasper targeting vector and native genomic DNA were cotransformed into yeast. The gene of interest is selectively cloned by recombination between the recombinogenic ends in the targeting vector and homologous regions in the genomic DNA. Here we demonstrate direct cloning of a stably integrated Hoxc8-LacZ-Ura3 reporter gene construct from a mouse embryo fibroblast cell line and single-copy genes from total human genomic DNA. The frequency of capture of the recombinant insert was 0.05-3% of transformants. In contrast to previous reports, we were able to clone genomic DNA directly with a vector containing yeast autonomous replicating sequences. This approach provides a powerful method with which to clone and modify genes precisely for functional analysis.

Animals↗

PubMed1999

Evolution of chordate hox gene clusters.

In this article, we consider the role of the Hox genes in chordate and vertebrate evolution from the viewpoints of molecular and developmental evolution. Models of Hox cluster duplication are considered with emphasis on a threefold duplication model. We also show that cluster duplication is consistent with a semiconservative model of duplication, where following duplication, one daughter cluster remains unmodified, while the other diverges and assumes a new architecture and presumably new functions. Evidence is reviewed, suggesting that Hox gene enhancers have played an important role in body plan evolution. Finally, we contrast the invertebrates and vertebrates in terms of genome and Hox cluster duplication which are present in the latter, but not the former. We question whether gene duplication has been important in vertebrates for the introduction of novel features such as limbs, a urogenital system, and specialized neuromuscular interactions.

Animals↗

PubMed1999

pPAC-ResQ: A yeast-bacterial shuttle vector for capturing inserts from P1 and PAC clones by recombinogenic targeted cloning.

We have developed a method to capture inserts from P1 and P1 artificial chromosome (PAC) clones into a yeast-bacteria shuttle vector by using recombinogenic targeting. We have engineered a vector, pPAC-ResQ, a derivative of pClasper, which was previously used to capture inserts from yeast artificial chromosome clones. pPAC-ResQ contains DNA fragments flanking the inserts in P1 and PAC vectors as recombinogenic ends. When linearized pPAC-ResQ vector and P1 or PAC DNA are cotransformed into yeast, recombination between the two leads to the transfer of inserts into pPAC-ResQ. pPAC-ResQ clones thus obtained can be further modified in yeast for functional analysis and shuttled to Escherichia coli to produce large quantities of cloned DNA. This approach provides a rapid method to modify P1/PAC clones for functional analysis.

Bacteria↗

PubMed1999

Ksp-cadherin gene promoter. II. Kidney-specific activity in transgenic mice.

Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a tissue-specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of tubular epithelial cells in the kidney. To determine the basis for tissue-specific expression of Ksp-cadherin in vivo, we evaluated the activity of the promoter in transgenic mice. Transgenic mice containing 3.3 kb of the mouse Ksp-cadherin promoter and an Escherichia coli lacZ reporter gene were generated by pronuclear microinjection. Assays of beta-galactosidase enzyme activity showed that the transgene was expressed exclusively in the kidney in both adult and developing mice. Within the kidney, the transgene was expressed in a subset of renal tubular epithelial cells that endogenously expressed Ksp-cadherin and that were identified as collecting ducts by colabeling with Dolichos biflorus agglutinin. In the developing metanephros, expression of the transgene in the branching ureteric bud correlated with the developmental expression of Ksp-cadherin. Identical patterns of expression were observed in multiple founder mice, indicating that kidney specificity was independent of transgene integration site. However, heterocellular expression was observed consistent with repeat-induced gene silencing. We conclude that the Ksp-cadherin gene promoter directs kidney-specific expression in vivo. Regulatory elements that are sufficient to recapitulate the tissue- and differentiation-specific expression of Ksp-cadherin in the renal collecting duct are located within 3.3 kb upstream to the transcriptional start site.

Aging↗

PubMed1998

Comparative studies on mammalian Hoxc8 early enhancer sequence reveal a baleen whale-specific deletion of a cis-acting element.

Variations in regulatory regions of developmental control genes have been implicated in the divergence of axial morphologies. To find potentially significant changes in cis-regulatory regions, we compared nucleotide sequences and activities of mammalian Hoxc8 early enhancers. The nucleotide sequence of the early enhancer region is extremely conserved among mammalian clades, with five previously described cis-acting elements, A-E, being invariant. However, a 4-bp deletion within element C of the Hoxc8 early enhancer sequence is observed in baleen whales. When assayed in transgenic mouse embryos, a baleen whale enhancer (unlike other mammalian enhancers) directs expression of the reporter gene to more posterior regions of the neural tube but fails to direct expression to posterior mesoderm. We suggest that regulation of Hoxc8 in baleen whales differs from other mammalian species and may be associated with variation in axial morphology.

Animals↗

PubMed1998

Recombinogenic targeting: a new approach to genomic analysis--a review.

Currently, recombinational cloning procedures based upon methods developed for yeast, Saccharomyces cerevisiae, are being exploited for targeted cloning and in-vivo modification of genomic clones. In this review, we will discuss the development of large-insert vectors, homologous recombination-based techniques for cloning and modification, and their application towards functional analysis of genes using transgenic mouse model systems.

Animals↗

PubMed1998

Modification of expression and cis-regulation of Hoxc8 in the evolution of diverged axial morphology.

Differential Hox gene expression between vertebrate species has been implicated in the divergence of axial morphology. To examine this relationship, we have compared expression and transcriptional regulation of Hoxc8 in chicken and mouse. In both species, expression of Hoxc8 in the paraxial mesoderm and neural tube is associated with midthoracic and brachial identities, respectively. During embryogenesis, there is a temporal delay in the activation of Hoxc8 in chicken compared with mouse. As a result, chicken Hoxc8 expression in the paraxial mesoderm is at a posterior axial level, extending over a smaller domain compared with mouse Hoxc8 expression. This finding is consistent with a shorter thoracic region in chicken compared with mouse. In addition, the chicken Hoxc8 early enhancer, differing from its mouse counterpart in only a few specific nucleotides, directs a reporter gene expression to a more posterior domain in transgenic mouse embryos. These findings are consistent with the concept that the diversification of axial morphology has been achieved through changes in cis-regulation of developmental control genes.

Animals↗

PubMed1998

Molecular evolution of Hox gene regulation: cloning and transgenic analysis of the lamprey HoxQ8 gene.

The mammalian Hox clusters arose by duplication of a primordial cluster. The duplication of Hox clusters created redundancy within cognate groups, allowing for change in function over time. The lamprey, Petromyzon marinus, occupies an intermediate position within the chordates, both in terms of morphologic complexity and possibly cluster number. To determine the extent of divergence among Hox genes after duplication events within vertebrates, we analyzed Hox genes belonging to cognate group 8. Here we report characterization of the HoxQ8 gene, which shows conservation with mammalian genes in its amino-terminal, homeobox and hexapeptide sequences, and in the position of its splice sites. A beta-galactosidase reporter gene was introduced in the HoxQ8 genomic region by targeted recombinational cloning using a yeast-bacteria shuttle vector, pClasper. These reporter gene constructs were tested for their ability to direct region-specific expression patterns in transgenic mouse embryos. Lamprey enhancers direct expression to posterior neural tube but not to mesoderm, suggesting conservation of neuronal enhancers. In the presence of the mouse heat shock promoter, lamprey enhancers could also direct expression to the posterior mesoderm suggesting that there has been some divergence in promoter function. Our results suggest that comparative studies on Hox gene structure and analysis of regulatory elements may provide insights into changes concomitant with Hox cluster duplications in the chordates.

Amino Acid Sequence↗

PubMed1996

Combinations of closely situated cis-acting elements determine tissue-specific patterns and anterior extent of early Hoxc8 expression.

We have used a transgene mutation approach to study how expression domains of Hoxc8 are established during mouse embryogenesis. A cis-regulatory region located 3 kb upstream from the Hoxc8 translational start site directs the early phase of expression. Four elements, termed A, B, C, and D, were previously shown to direct expression to the neural tube. Here we report that a fifth element, E, located immediately downstream of D directs expression to mesoderm in combination with the other four elements. These elements are interdependent and partially redundant. Different combinations of elements determine expression in different posterior regions of the embryo. Neural tube expression is determined minimally by ABC, ABD, or ACD; somite expression by ACDE; and lateral plate mesoderm expression by DE. Neural tube and lateral plate mesoderm enhancers can be separated, but independent somite expression has not been achieved. Furthermore, mutations within these elements result in posteriorization of the reporter gene expression. Thus, the anterior extent of expression is determined by the combined action of these elements. We propose that the early phase of Hoxc8 expression is directed by two separate mechanisms: one that determines tissue specificity and another that determines anterior extent of expression.

Animals↗

PubMed1996

A long-range regulatory element of Hoxc8 identified by using the pClasper vector.

Hox genes are located in highly conserved clusters. The significance of this organization is unclear, but one possibility is that regulatory regions for individual genes are dispersed throughout the cluster and shared with other Hox genes. This hypothesis is supported by studies on several Hox genes in which even large genomic regions immediately surrounding the gene fail to direct the complete expression pattern in transgenic mice. In particular, previous studies have identified proximal regulatory regions that are primarily responsible for early phases of mouse Hoxc8 expression. To locate additional regulatory regions governing expression during the later periods of development, a yeast homologous recombination-based strategy utilizing the pClasper vector was employed. Using homologous recombination into pClasper, we cloned a 27-kb region around the Hoxc8 gene from a yeast artificial chromosome. A reporter gene was introduced into the coding region of the isolated gene by homologous recombination in yeast. This large fragment recapitulates critical aspects of Hoxc8 expression in transgenic mice. We show that the regulatory elements that maintain the anterior boundaries of expression in the neural tube and paraxial mesoderm are located between 11 and 19 kb downstream of the gene.

Animals↗

PubMed1996

Conserved regulatory element involved in the early onset of Hoxb6 gene expression.

We have identified a 338 bp DNA fragment, the lateral plate mesoderm (LPM) enhancer, that is highly conserved between mouse and human. The LPM enhancer directs gene expression into the posterior lateral plate mesoderm and hindgut endoderm at early stages of development. By reporter gene analysis in transgenic mice, we demonstrate that both mouse and human DNA sequences possess similar enhancer activity. The expression patterns of the transgene and Hoxb6 during early stages of mouse development are identical, suggesting that the LPM enhancer is involved in the initial activation of Hoxb6 gene expression in posterior regions of mammalian embryos.

Animals↗

PubMed1996

Separate cis-acting elements determine the expression of mouse Dbx gene in multiple spatial domains of the central nervous system.

Dbx, a divergent homeobox gene, is expressed in a regionally restricted pattern in the developing mouse central nervous system (CNS). In order to understand its spatial regulation, we have isolated a cis-regulatory region using a reporter gene analytical approach in transgenic mice. A 5.7 kb DNA fragment that contains the transcriptional start site of the Dbx gene is sufficient to direct the expression of the transgene to various domains of the CNS in a temporally regulated fashion. The transgene expression can be detected between 9.5 and 15.5 days post coitum in embryos in the fore-, mid- and hindbrain and spinal cord in regions where the endogenous gene is expressed. Additionally, transgene expression can also be detected in the oculomotor nerve (cranial nerve III). The expression of the transgene closely resembles that of the Dbx gene with minor, but interesting differences. These results suggest that major cis-acting elements reside within a 5.7 kb DNA fragment located 5' of the Dbx gene. Further deletion analysis shows that at least two independently regulated elements are present within this DNA fragment: an element that directs expression to the brain and spinal cord and a second element that directs expression to the oculomotor nerve.

Amino Acid Sequence↗

PubMed1995

Regulation of Hoxc-8 during mouse embryonic development: identification and characterization of critical elements involved in early neural tube expression.

We have characterized cis-acting elements that direct the early phase of Hoxc-8 expression using reporter gene analysis in transgenic mice. By deletion we show that a 135 bp DNA fragment, located approximately 3 kb upstream of the coding region of Hoxc-8, is capable of directing posterior neural tube expression. This early neural tube (ENT) enhancer consists of four separate elements, designated A, B, C and D, whose nucleotide sequences are similar to binding sites of known transcription factors. Nucleotide substitutions suggest that element A is an essential component of the ENT enhancer. However element A by itself is incapable of directing neural tube expression. This element requires interactions at any two of the other three elements, B, C or D. Thus, the components of the ENT enhancer direct neural tube expression in an interdependent manner. We propose that Hoxc-8 is activated in the neural tube by combinatorial interactions among several proteins acting within a small region. Our transgenic analyses provide a means to identify transcription factors that regulate Hoxc-8 expression during embryogenesis.

Animals↗

PubMed1992

Repression of the beta-amyloid gene in a Hox-3.1-producing cell line.

Mammalian homeobox genes are widely expressed in the developing central nervous system and are postulated to control developmental processes by regulating gene expression at the transcriptional level. In vitro studies have identified consensus DNA sequences that contain an ATTA core as sites for interaction with homeodomain proteins. Such elements have been found in the upstream regulatory region of the gene encoding beta-amyloid precursor protein, which is associated with the neurological disorder Alzheimer disease. As the beta-amyloid precursor protein gene is also expressed in the developing central nervous system and appears to play a role in cellular regulatory processes, we have examined the possibility that a homeobox gene product can regulate its transcription. We demonstrate by Northern blot analyses and transfection experiments that the expression of the beta-amyloid precursor protein gene is decreased in cultured cells expressing the mouse homeobox gene Hox-3.1.

Amyloid beta-Protein Precursor↗

PubMed1991

Homeobox genes in mouse development.

Following the discovery of the homeobox as a conserved sequence in developmentally important genes of Drosophila, a plethora of such sequences have been identified in evolutionarily distant organisms. Among mammals, the mouse homeobox genes have been studied most intensively with a hope of deciphering basic mechanisms of embryonic development. The genomic arrangement of many mouse homeobox genes is similar to the organization of the Drosophila genes, suggesting that they arose as a consequence of gene duplication and divergence from a primordial cluster during evolution. Homeobox genes encode proteins that may form a part of the autoregulatory and transregulatory network specifying positional value in the embryo. Supporting this view, the more diverged members of this growing family function as transcription factors, some of which regulate the expression of tissue-specific genes. Mouse homeobox genes are expressed during embryonic development in a spatially restricted manner and alterations in their expression pattern can disrupt embryonic development. The implications of these findings will be discussed in the context of the role of homeobox genes in the embryonic development of Drosophila and other organisms.

Amino Acid Sequence↗

PubMed1983

A DNA polymerase activity associated with the skeletal framework of the plasma membranes of a rat hepatoma.

A DNA polymerase activity has been identified in the plasma membranes isolated from a rat hepatoma (the Zajdela Ascitic Hepatoma). The enzyme activity was found specifically associated with the detergent insoluble skeleton of the plasma membranes from which it cannot be dissociated by conventional methods. The properties of the enzyme are indistinguishable from those of DNA polymerase-alpha.

Animals↗