Biomedical subjects

F H Ruddle

Publications and source records attributed to F H Ruddle.

At least 37 records · Page 2Linked to original sources

PubMed1997

Phylogenetic reconstruction of vertebrate Hox cluster duplications.

In vertebrates and the cephalochordate, amphioxus, the closest vertebrate relative, Hox genes are linked in a single cluster. Accompanying the emergence of higher vertebrates, the Hox gene cluster duplicated in either a single step or multiple steps, resulting in the four-cluster state present in teleosts and tetrapods. Mammalian Hox clusters (designated A, B, C, and D) extend over 100 kb and are located on four different chromosomes. Reconstructing the history of the duplications and its relation to vertebrate evolution has been problematic due to the lack of alignable sequence information. In this study, the problem was approached by conducting a statistical analysis of sequences from the fibrillar-type collagens (I, II, III, and IV), genes closely linked to each Hox cluster which likely share the same duplication history as the Hox genes. We find statistical support for the hypothesis that the cluster duplication occurred as multiple distinct events and that the four-cluster situation arose by a three-step sequential process.

Animals↗

PubMed1996

Genomic analysis of a new mammalian distal-less gene: Dlx7.

We have cloned a new Dlx gene (Dlx7) from human and mouse that may represent the mammalian orthologue of the newt gene NvHBox-5. The homeodomains of these genes are highly similar to all other vertebrate Dlx genes, and regions of similarity also exist between mammalian Dlx7 and a subset of vertebrate Dlx genes downstream of the homeodomain. The sequence divergence between human and mouse Dlx7 in these regions is greater than that predicted from comparisons of other vertebrate Dlx genes, however, and there is little sequence similarity upstream of the homeodomain both between these two genes and with other Dlx genes. We present evidence for alternative splicing of mouse Dlx7 upstream of the homeodomain that may account for some of this divergence. We have mapped human DLX7 distal to the 5' end of the HOXB cluster at an estimated distance of between 1 and 2 Mb by FISH. Both the human and the mouse Dlx7 are shown to be closely linked to Dlx3 in a convergently transcribed orientation. These mapping results support the possibility that vertebrate distal-less genes have been duplicated in concert with the Hox clusters.

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

The evolution of the vertebrate Dlx gene family.

The vertebrate Dlx gene family consists of homeobox-containing transcription factors distributed in pairs on the same chromosomes as the Hox genes. To investigate the evolutionary history of Dlx genes, we have cloned five new zebrafish family members and have provided additional sequence information for two mouse genes. Phylogenetic analyses of Dlx gene sequences considered in the context of their chromosomal arrangements suggest that an initial tandem duplication produced a linked pair of Dlx genes after the divergence of chordates and arthropods but prior to the divergence of tunicates and vertebrates. This pair of Dlx genes was then duplicated in the chromosomal events that led to the four clusters of Hox genes characteristic of bony fish and tetrapods. It is possible that a pair of Dlx genes linked to the Hoxc cluster has been lost from mammals. We were unable to distinguish between independent duplication and retention of the ancestral state of bony vertebrates to explain the presence of a greater number of Dlx genes in zebrafish than mammals. Determination of the linkage relationship of these additional zebrafish Dlx genes to Hox clusters should help resolve this issue.

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

Characterization and sequence analysis of the human homeobox-containing gene GBX2.

Polymerase chain reaction (PCR) was used to amplify portion of homeobox genes present in a human 11-week fetal brain cDNA library. One of these PCR products was determined by sequencing to be the Gastrulation and brain specific-2 gene (GBX2). Screening this human fetal brain cDNA library with probes specific for GBX2 led to the identification of a 2151-bp cDNA clone. The nucleotide sequence of the cDNA clone encodes for a protein of 347 amino acid residues. The amino acid sequence of the GBX2 homeodomain is identical (100%) to the that of homologous gene, Gbx2, expressed in the developing mouse embryo and virtually identical (97%) to a gene expressed in the developing chicken embryo, CHox7. The 5' end of the GBX2 gene contains a CpG island in the untranslated region and a trinucleotide (CCG)8 repeat in the coding region. The amino-terminal end of the GBX2 protein is proline-rich, with 30 proline residues in one stretch of 120 amino acids. A single 2.2-kb transcript was detected by Northern analysis in the developing human CNS as well as in other tissues. The human genomic clone for GBX2 was also isolated, characterized, and mapped to 2q36(d)-q37 by somatic cell hybrid analysis and fluorescence in situ hybridization. These studies provide a framework for designing future experiments that are needed to determine the functional significance of this gene in CNS development.

Amino Acid Sequence↗

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

Spatial and temporal regulation of a lacZ reporter transgene in a binary transgenic mouse system.

The transgenic mouse system is a powerful tool for the study of gene function. However, when the analysis involves genes that are critical for the normal developmental process, the usefulness of transgenic mouse systems is limited (for review see Hanahan, 1989; Westphal and Gruss, 1989; Byrne et al., 1991). This is due to potential transgene interference with development in case of ectopic or high level expression. As a result, establishing permanent transgenic mouse lines expressing these types of genes has proven difficult. To circumvent these difficulties, a binary transgenic mouse system has been established, termed the Multiplex System (Byrne and Ruddle, 1989). This is a two-tiered gene activation system in which expression of the gene of interest occurs only in offspring carrying transgenes encoding both components: transactivator and transresponder. Transactivator lines contain the gene encoding the VP16 protein of herpes simplex virus. Transresponder lines harbour the gene of interest linked to the IE promoter which includes recognition sequences for the VP16 transactivator. Previously, the inducibility of a chloramphenicol acetyltransferase reporter gene in newborn offspring that carried both a transactivator and transresponder transgene (Byrne and Ruddle, 1989) has been shown. Moreover, it has been demonstrated that expression of the VP16 protein was not detrimental to development and that transactivation appeared to be tissue specific. Here, the potential of the system for the expression of transgenes in early mouse embryogenesis was examined, using the Escherichia coli beta-galactosidase gene as a reporter in the transresponder mouse strain. To direct expression of VP16, the murine Hoxc-8 promoter, which is known to be active during early development, was used. Embryos from crosses of transactivators to transresponders were isolated at different stages of development and stained for beta-galactosidase activity. Transactivation, as demonstrated by strong beta-galactosidase staining, could be detected as early as eight days of development. At all stages examined, the pattern of lacZ transresponder gene expression accurately reflected the activity of the Hoxc-8 promoter controlling VP16 expression. It is demonstrated that the Multiplex System can be used to express transresponder transgenes in a spatially and temporally defined manner in multiple cell types early during mouse embryogenesis.

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

A new vector for recombination-based cloning of large DNA fragments from yeast artificial chromosomes.

The functional analysis of genes frequently requires manipulation of large genomic regions embedded in yeast artificial chromosomes (YACs). We have designed a yeast-bacteria shuttle vector, pClasper, that can be used to clone specific regions of interest from YACs by homologous recombination. The important feature of pClasper is the presence of the mini-F factor replicon. This leads to a significant increase in the size of the plasmid inserts that can be maintained in bacteria after cloning by homologous recombination in yeast. The utility of this vector lies in its ability to maintain large fragments in bacteria and yeast, allowing for mutagenesis in yeast and simplified preparation of plasmid DNA in bacteria. Using PCR-generated recombinogenic fragments in pClasper we cloned a 27 kb region from a YAC containing the Hoxc cluster and a 130 kb region containing the entire Hoxb cluster. No rearrangements were seen when the recombinants in the shuttle vector were transferred to bacteria. We outline the potential uses of pClasper for functional studies of large genomic regions by transgenic and other analyses.

Base Sequence↗

PubMed1995

Human HOXB cluster and the nerve growth factor receptor gene: comparison with an orthologous chromosomal domain in mouse.

The structural organization and nucleotide sequence similarity of mammalian Antennapedia-class homeobox genes support the view that the four homeobox clusters (HOXA, B, C, and D on human chromosomes 7, 17, 12, and 2, respectively) arose through a combination of gene duplication and divergence to form a cluster, followed by several cluster duplications. The duplication events that gave rise to the four clusters appear to have involved chromosomal domains extending well beyond the borders of the clusters in either direction. This evidence arises from the observation that many genes closely linked to the homeobox clusters on different chromosomes show sequence similarity. Here, we present a continuation of physical mapping studies to determine the extent and organization of the duplicated regions surrounding the four homeobox clusters in human. Southern blots prepared from pulsed-field gels of human DNA were probed with cloned segments of human HOXB genes and the nerve growth factor receptor (NGFR) gene on chromosome 17q21-q22. Restriction enzyme analysis revealed the close physical linkage of these genes within 100 kb. Two yeast artificial chromosomes (YACs), 220 and 380 kb in size, were isolated using oligonucleotide primers specific for NGFR. Both YACs contained the entire HOXB cluster. Restriction mapping of the clones indicated that the distance separating these loci could not be greater than 50 kb. This result confirms and extends previous information on the proximity of these genes as determined by genetic linkage analysis and closely parallels the orthologous loci in the mouse.

Animals↗

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↗

PubMed1995

Dlx and other homeobox genes in the morphological development of the dentition.

The dentition is a segmental system whose evolution and morphology bears analogy to the evolution of segmentation in the vertebral column and limb. Combinatorial expression of members of the large "Hox" class of homeobox regulatory genes has been shown to play an important role in positional specification in these skeletal systems. This raises the possibility that homeobox genes are also used for positional specification in the dentition, and several homeobox genes are known to be expressed in developing teeth. To identify additional dentally expressed homeobox genes, cDNA from from murine tooth germs at 9.5, 14.5, and 17.5 days gestational age was amplified by PCR using sets of degenerate primers to the homeodomains of 18 different classes of homeobox genes. Amplification products were cloned and sequenced and compared to known gene sequences. To date this approach has confirmed the presence of Msx1, Msx2, Dlx1, and Dlx2, and identified several other homeobox genes not previously known to be expressed in teeth: Dbx, MHox, and Mox2A, plus an a additional Dlx gene, Dlx7. The Msx and Dlx genes are the best current candidates for a combinatorial mechanism that controls the differentiation of structures within and between teeth, and perhaps also the evolution of those structures.

Amino Acid Sequence↗

PubMed1994

Distal-less and other homeobox genes in the development of the dentition.

The mammalian tooth develops through an interaction between two tissue layers of different embryologic origin. A number of transcription factors and as well as two members of the Msx class of homeobox genes have been shown to be involved in the histogenesis of the mammalian tooth. This raised the possibility that other homeobox genes might be involved in dental morphogenesis. We have amplified mouse tooth germ cDNA from three different gestational ages by the polymerase chain reaction with degenerate primers for 18 classes of homeobox genes. Members of several classes have been isolated, including the Msx genes, two Dlx genes, and the Dbx, MHox, Mox2A genes. One of the Dlx genes, Dlx-7, had not previously been reported in mammals, and some details are presented of its cDNA sequence. This work plus that of other investigators has shown that at least six Dlx genes are expressed in developing teeth or in first branchial arches, suggesting the possibility that these genes are involved in specifying complexity within or between teeth. The screening approach with degenerate primers is a successful way to identify new as well as previously known regulatory genes expressed in developing tooth embryos.

Amino Acid Sequence↗

PubMed1994

A DNA-binding activity, TRAC, specific for the TRA element of the transferrin receptor gene copurifies with the Ku autoantigen.

We have previously described purification and characterization of a nuclear protein, TREF, which interacts specifically with the transcriptional control element, TRA, of the human transferrin receptor (TR) gene. In this report we show that TREF can be separated into two functionally distinct DNA-binding activities. The first DNA-binding activity (TRAC) is highly specific for the 8-bp element TRA and the related Escherichia coli cAMP receptor binding site. This motif is homologous to the phorbol 12-tetradecanoate 13-acetate- and cAMP-responsive elements of eukaryotic genes and the regulatory proximal sequence elements of the U1 small nuclear RNA gene and is also present in the promoter of the Drosophila melanogaster yolk protein factor 1 gene. In striking contrast, the second activity exhibits high affinity for the ends of double-stranded DNA in a sequence-unspecific manner and is attributable to the heterodimeric Ku autoantigen. Notably, transcription of Ku is induced during mid-late G0/G1 with kinetics similar to the TR gene. Ku is a highly abundant nuclear protein possessing nonspecific affinity for the ends of DNA, whose biological role remains to be elucidated. A transcriptional role for this protein has been proposed, however, on the basis of studies attributing DNA sequence-specific binding activity, notably for TRA-like sequences described above, directly to the Ku heterodimer. The observation that Ku-mediated nonspecific DNA-binding activity copurifies with the TRA-specific activity, TRAC, clearly has implications for these and related studies. The unusual properties of TRAC activity and its relationship, if any, with the enigmatic Ku protein, are discussed.

Antigens, Nuclear↗

PubMed1994

Identification of the murine Hox-c12 and Hox-c13 homeoboxes on yeast artificial chromosomes.

The structure of the Antennapedia-class homeobox gene clusters is highly conserved in human and mouse. Of all the human homeobox genes known, only HOX-C12 and HOX-C13 have not also been identified in mouse. These two genes are located at the extreme 5' end of the human HOX-C cluster and are predicted to be located in a similar position on the murine Hox-c cluster. We have isolated two yeast artificial chromosomes (YACs) containing the murine Hox-c cluster. We designed primers specific to the human HOX-C12 and -C13 homeoboxes and used the polymerase chain reaction and sequencing to identify the homeobox of the murine orthologs of these two genes on one of the YACs. Only the YAC containing the 5' end of the Hox-c cluster contained these sequences. Thus, we have identified two mouse homeobox genes not previously reported and established linkage of these genes to the 5' end of the mouse Hox-c cluster.

Animals↗

PubMed1994

Embryonic expression pattern of amyloid protein precursor suggests a role in differentiation of specific subsets of neurons.

Immunohistochemical analysis revealed the temporal and spatial expression pattern of the amyloid protein precursor (APP) during the development of the mouse embryo. APP was first detected at day 9.5 of gestation in motor neurons of the hind brain and the spinal cord. APP proteins were also evident in cells of the floor plate, and in neurons of the cranial, dorsal root, and sympathetic ganglia shortly after their formation. Except for floor plate cells, APP expression was restricted to differentiated neurons. Comparison with the expression of microtubule-associated protein 2 (MAP-2), a marker for neurodifferentiation, showed that APP is expressed on a subset of differentiated neurons. APP can also serve as an early marker for the developing nuclei of the hind brain. The onset of APP expression in neurons appeared to be correlated with axonal outgrowth, whereas later expression of APP may be associated with functional specialization in the developing nervous system.

Amyloid beta-Protein Precursor↗

PubMed1994

Chromosomal assignment of three novel mouse genes expressed in testicular cells.

The chromosomal positions of three genes that are selectively expressed in mouse testis cells have been identified. These genes include (i) TAZ83, which codes for an early- to mid-pachytene germ cell stage-expressed, cysteine-rich transmembrane protein (cyritestin) with homologies to various snake toxins and guinea pig sperm-egg fusion proteins; (ii) TNZ1, which is expressed in neonatal Leydig cells; and (iii) TAZ4, a testis-specific gene isolated by immunoscreening with antiserum raised against Sertoli cell membranes. Our experimental data, derived from chromosomal in situ hybridizations and RFLP studies of genetic backcrosses, indicate that (i) the TAZ83 (cyritestin) gene maps to chromosome 8, band A2, near the Plat locus; (ii) TNZ1 is located in the proximal region of chromosome 11; and (iii) TAZ4 is located at band D in the distal portion of chromosome 11, near the Hlr1 locus, with a related sequence, TAZ4-rs1, in the proximal part of chromosome 1.

Animals↗