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A Fainsod

Publications and source records attributed to A Fainsod.

At least 37 records · Page 2Linked to original sources
PubMed1993

Isolation and characterization of target sequences of the chicken CdxA homeobox gene.

The DNA binding specificity of the chicken homeodomain protein CDXA was studied. Using a CDXA-glutathione-S-transferase fusion protein, DNA fragments containing the binding site for this protein were isolated. The sources of DNA were oligonucleotides with random sequence and chicken genomic DNA. The DNA fragments isolated were sequenced and tested in DNA binding assays. Sequencing revealed that most DNA fragments are AT rich which is a common feature of homeodomain binding sites. By electrophoretic mobility shift assays it was shown that the different target sequences isolated bind to the CDXA protein with different affinities. The specific sequences bound by the CDXA protein in the genomic fragments isolated, were determined by DNase I footprinting. From the footprinted sequences, the CDXA consensus binding site was determined. The CDXA protein binds the consensus sequence A, A/T, T, A/T, A, T, A/G. The CAUDAL binding site in the ftz promoter is also included in this consensus sequence. When tested, some of the genomic target sequences were capable of enhancing the transcriptional activity of reporter plasmids when introduced into CDXA expressing cells. This study determined the DNA sequence specificity of the CDXA protein and it also shows that this protein can further activate transcription in cells in culture.

Amino Acid Sequence↗
PubMed1993

The chicken CdxA homeobox gene and axial positioning during gastrulation.

The chicken homebox containing gene, CdxA (formerly CHox-cad), was previously shown to be expressed during gastrulation. Localization of CdxA transcripts by in situ hybridization to tissue sections revealed that, during gastrulation, expression of this gene exhibits a posterior localization along the primitive streak. The transcripts are localized to epiblast cells in the vicinity of the primitive streak, to cells of the primitive streak itself and in the definitive endoderm as it replaces the hypoblast. In order to study in greater detail the pattern of expression of the CdxA gene during gastrulation, we expressed the full-length CdxA protein as a fusion protein in E. coli and generated monoclonal antibodies against it. Chicken embryos at different stages of gastrulation were processed for whole-mount immunohistochemical localization of the protein using anti-CdxA antibodies. Once the pattern of expression in the whole embryo was determined, the same embryos were sectioned to determine the identity of the cells expressing the CdxA protein. Detailed analysis of the CdxA protein in embryos, from the onset of primitive streak formation to the beginning of the tail bud stage (stages 2 to 10), has shown different patterns of expression during primitive streak elongation and regression. The CdxA protein is initially detected at the posterior marginal zone and the expression moves rostrally into the primitive streak during mid-streak stages. As the primitive streak elongates, the CdxA stripe of expression moves anteriorly. By definitive streak stages, the CdxA stripe of expression delineates a position along the anterior-posterior axis in the primitive streak. CdxA, like its Drosophila homologue cad, is expressed during gastrulation in a stripe localized to the posterior region of the embryo. These observations suggest that CdxA as a homebox gene may be part of a regulatory network coupled to axial determination during gastrulation in the early chick embryo.

Animals↗
PubMed1991

Genomic organization and expression during embryogenesis of the chicken CR1 repeat.

CR1 is one of the middle repetitive sequence elements present in the chicken genome. One such repetitive element (GG1-CR1) was found upstream of the chicken CHox E homeobox gene. Sequencing of GG1-CR1 demonstrated that it is one of the longest CR1 elements analyzed. Detailed comparison of all the CR1 sequences published has revealed three subfamilies of CR1 repeats containing various parts of the consensus sequence. We prepared DNA fragments from GG1-CR1 and used them to probe Southern blots and genomic and cDNA library lifts. The results confirm the division of CR1 into three subelements, two of which occur independently in many places in the genome. Northern blot analysis of the CR1 to chicken embryo RNA showed that the CR1 repeat can be part of poly(A)+ transcripts. These results suggest that the CR1 can be transcribed by readthrough from the promoter of the neighboring gene without detrimental effects on the expression of the gene itself. The level of CR1 containing transcripts rises during the first 5 days of embryonic development and then decreases.

Animals↗
PubMed1991

CHox E, a chicken homeogene of the H2.0 type exhibits dorso-ventral restriction in the proliferating region of the spinal cord.

CHox E is a novel chicken homeogene that belongs to the H2.0 family of homeodomains. Its homeobox sequence is interrupted by an intron between amino acids 44 and 45. Expression of CHox E during embryogenesis is localized to the central nervous system. The anterior boundary of CHox E expression can initially be localized to rhombomere number 1, later in development this boundary reaches up to the rhombencephalic isthmus. CHox E expression in the spinal cord localizes dorso-ventrally to the dorsal half of the basal plate. CHox E expression is always restricted to the proliferating region, the ventricular zone. As the ventricular zone becomes restricted laterally, so does the CHox E expressing region. Once this region of the ventricular zone ceases to exist, CHox E specific transcripts become undetectable. The site and time of CHox E expression suggest a very early function in the differentiation of the cells derived from that region of the ventricular zone.

Amino Acid Sequence↗
PubMed1991

A chicken caudal homologue, CHox-cad, is expressed in the epiblast with posterior localization and in the early endodermal lineage.

CHox-cad is a chicken homeobox gene whose homeodomain is homologous to the Drosophila caudal and the murine Cdx1 genes. Based on sequence analysis of a 2.5 kb CHox-cad cDNA clone, we deduced that the primary translation product consists of 248 amino acids. Comparison between the cDNA and genomic clones revealed the presence of an intron within the CHox-cad homeodomain between amino acids 44 and 45. The onset of CHox-cad transcription correlates temporarily with the beginning of gastrulation. During primitive streak stages CHox-cad exhibits a caudally localized pattern of expression restricted to the epiblast and the primitive streak. At these stages, CHox-cad transcripts can also be detected in the definitive endoderm cells. Later in embryogenesis CHox-cad is expressed in the epithelial lining of the embryonic gut and yolk sac. After four days of chicken development, no CHox-cad transcripts could be detected. The early CHox-cad posterior expression in the germ layer undergoing gastrulation and its continuous expression in the early endodermal lineage raise the possibility of CHox-cad involvement in the establishment of the definitive endoderm.

Amino Acid Sequence↗
PubMed1990

Isolation and characterization of the Drosophila nuclear envelope otefin cDNA.

We have recently identified and characterized a 53-kDa inner nuclear membrane-associated protein in Drosophila and termed it otefin. Here we report the isolation and characterization of cDNA and genomic clones of the otefin gene. Based on sequence analysis, we deduced that the primary translation product has a calculated mass of 45 kDa, contains many serine and threonine residues, and is mostly hydrophilic. However, in the carboxyl terminus, there is a hydrophobic region which may serve as a membrane anchoring domain. RNA blot analysis indicated that the otefin gene codes for a single poly(A+) transcript of 1.6 kilobases and that relatively large amounts of this transcript are present during developmental stages in which many nuclear divisions occur. Polyclonal antibodies raised against the cDNA translation product react with a 58-kDa mammalian nuclear envelope protein, demonstrating evolutionary conservation.

Amino Acid Sequence↗
PubMed1990

Cloning, characterization, and expression in Escherichia coli of the gene coding for the CpG DNA methylase from Spiroplasma sp. strain MQ1(M.SssI).

We describe here the cloning, characterization and expression in E. coli of the gene coding for a DNA methylase from Spiroplasma sp. strain MQ1 (M.SssI). This enzyme methylates completely and exclusively CpG sequences. The Spiroplasma gene was transcribed in E. coli using its own promoter. Translation of the entire message required the use of an opal suppressor, suggesting that UGA triplets code for tryptophan in Spiroplasma. Sequence analysis of the gene revealed several UGA triplets, in a 1158 bp long open reading frame. The deduced amino acid sequence revealed in M.SssI all common domains characteristic of bacterial cytosine DNA methylases. The putative sequence recognition domain of M.SssI showed no obvious similarities with that of the mouse DNA methylase, in spite of their common sequence specificity. The cloned enzyme methylated exclusively CpG sequences both in vivo and in vitro. In contrast to the mammalian enzyme which is primarily a maintenance methylase, M.SssI displayed de novo methylase activity, characteristic of prokaryotic cytosine DNA methylases.

Amino Acid Sequence↗
PubMed1990

Molecular analysis of the Drosophila nuclear lamin gene.

A complete nucleotide sequence of a 4.2-kb genomic fragment containing the Drosophila lamin gene and flanking sequences is presented. Primer extension experiments and sequence analysis revealed that transcription starts from a single promoter. The lamin maternal 2.8-kb transcript and the 3.0-kb zygotic transcript are generated from two alternative polyadenylation sites. The gene contains four exons. The first intron is 7 bp upstream of the first AUG site. The two other introns are located within the alpha-helical rod domain of the protein: one in coil 1B in the 42-amino-acid domain that is absent in vertebrate cytoplasmic intermediate filament proteins and the other in coil 2 at a position different from intron positions within the vertebrate intermediate filament genes. Together with the sequence homology analysis, the data suggest either that the lamin gene was the ancestral gene of intermediate filament genes or that the lamin gene diverged from other intermediate filament genes early in evolution.

Amino Acid Sequence↗
PubMed1989

A chicken homeo box gene with developmentally regulated expression.

Several homeo box sequences were isolated from a chicken (Gallus domesticus) genomic library. One phage, lambda GG7, was chosen for further study and the single homeo box sequence it contains was characterized. The sequence of the homeo box revealed it to be a distant member of the Antp class of homeo boxes. Expression of the CHox7 gene results in four transcripts, 1.4, 1.9, 2.4 and 3.5 kb in size. Transcript accumulation of the CHox7 gene peaks twice during embryogenesis, once with the 1.4 kb transcript during early somitogenesis and the second time with the 1.9 kb transcript during organogenesis.

Amino Acid Sequence↗
PubMed1989

The chicken homeo box genes CHox1 and CHox3: cloning, sequencing and expression during embryogenesis.

Several Drosophila genes involved in the control of segmentation and segment identity share a 183-bp conserved sequence termed homeo box. Homeo box sequences have been detected and cloned from the genomes of insects like Drosophila to vertebrates such as mouse and man. Two chicken homeo box genes CHox1 and CHox3, are described. Cloning of the CHox1 and CHox3 homeo boxes was performed using Drosophila and murine homeo box sequences as probes under low-stringency conditions. Analysis of both chicken homeo box sequences revealed them to be homeo boxes that have diverged from the Antennapedia class with homologies to homeo boxes of other organisms in the range of 75-42% at the nucleotide level and 69-41% at the protein level. Analysis of CHox3 expression during early embryo development showed that the gene codes for five transcripts 1.3, 1.9, 2.6, 5.6 and 7.9 kb in size. Three of the transcripts (1.3, 1.9 and 5.6 kb) are also recognized by a flanking non-homeo box containing probe. The levels of the different transcripts changed during the first five days of development. The most abundant transcripts (1.3 and 1.9 kb) are already present at the time the egg is laid. Their transcription peaks at day 1 of incubation and then decreases. The CHox1 transcripts are present at very low levels between days 2.5 and 4 of development. These two chicken genes represent bona fide Hox genes in a branch of vertebrates that evolved parallel to mammals.

Amino Acid Sequence↗
PubMed1987

Expression of the murine homeo box gene Hox 1.5 during embryogenesis.

The spatial pattern of expression of the murine homeo box-containing gene Hox 1.5 was studied during embryogenesis. In situ hybridization of single-stranded RNA probes to mouse embryo sections revealed a specific spatial distribution of the Hox 1.5 transcripts in mouse embryos 8.5 to 12.5 days postcoitum (p.c.). Analysis of mouse embryos 8.5 days p.c. showed that the gene is expressed in a spatially restricted manner. Expression appears to be limited to the central nervous system with an anterior boundary in the hindbrain and extending posteriorly through caudal regions of the spinal cord. The same spatial pattern of expression was observed in embryos 9.5 to 12.5 days p.c. These results show that the murine Hox 1.5 gene is expressed in a spatially restricted manner during embryonic development similar to the patterns observed in Drosophila homeotic genes.

Animals↗
PubMed1987

Sequence analysis of the murine Hox-2.2, -2.3, and -2.4 homeo boxes: evolutionary and structural comparisons.

We have determined the nucleotide sequences and deduced the amino acid sequences of three tandemly arranged murine boxes of the Hox-2 homeo box gene complex on mouse chromosome 11 (Hox-2.2, -2.3, and -2.4). The type and position of differences with other sequenced homeo boxes were analyzed. Hox-2.2 is nearly identical with its cognate human homeo box Hu-2. Hox-2.3 shares 59 of 61 amino acids with the Antennapedia homeo domain of Drosophila and the MM-3 homeo domain of Xenopus and shows 60 of 61 amino acid identity with human HuC1. Hox-2.3, MM-3, and HuC1 also share a stretch of six glutamic acid residues followed by a stop codon 15-20 amino acids 3' of the homeo domain. Hox-2.4 is relatively divergent from most of the other homeo boxes sequenced to date; however, it matches the Hox-3.1 murine homeo domain at 60 of 61 positions. Sequence comparisons with other murine homeo domains, together with previous studies of their genomic organization and chromosomal location, provide support for the hypothesis of a large-scale duplication resulting in the two major murine homeo box gene complexes Hox-1 and Hox-2.

Amino Acid Sequence↗
PubMed1987

Homeo box genes in murine development.

Considerable information has accumulated on mouse homeo box gene organization and expression. Homeo box genes are expressed in a wide variety of tissues, developmental stages, and cell lines. How can this be interpreted in view of the relationship of these genes to Drosophila morphogenetic loci? One view is that homeo box genes control determinative decisions by modulating transcription of as yet unidentified target genes. Proponents of this view are faced with two tasks: to identify developmental processes that are controlled by homeo box genes, and to identify the target genes that mediate this control. Such target genes might be identified on the basis of in vitro homeo domain-DNA interactions. Candidate morphogenetic processes might be identified on the basis of the observed patterns of homeo box gene expression. It must be stressed that finding expression in a given tissue in no way demonstrates that the expression is necessary for the determination of that tissue. The role of Drosophila homeo box genes in determinative decisions is based upon analysis of mutants to demonstrate that the pattern of homeo box gene expression determines the morphogenetic outcome. To test whether the expression of a mouse homeo box gene is involved in a determinative decision, one must disrupt the normal pattern of expression of that gene and observe the resulting morphogenetic effect. In mouse this can be approached by looking for allelism with known morphogenetic loci, by isolating mutants in homeo box genes through large-scale mutagenesis screens, or by introducing altered homeo box genes into transgenic mice. One of the most intriguing possibilities is that homeo box genes are involved in regional specification along the anteroposterior axis. In situ hybridization and Northern blot analysis have demonstrated that at least four different homeo box genes display distinct regional patterns of expression along the anteroposterior axis of the developing CNS. The expression of each of these genes has a unique anterior boundary from which expression extends posteriorly within the CNS. Hox 1.5 expression has an anterior boundary within the hindbrain just posterior to the pontine flexure. The anterior boundary of Hox 2.1 expression lies more posteriorly within the medulla of the hindbrain. Weak expression of Hox 2.5 is detected in the spinal cord just posterior to the first cervical vertebra, and maximal expression is found posterior to the second cervical vertebra.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Cloning of a human S-phase cell cycle gene: use of transient expression for screening.

We report here the cloning of a human cell cycle gene capable of complementing a temperature-sensitive (ts) S-phase cell cycle mutation in a Chinese hamster cell line. Cloning was performed as follows. A human genomic library in phage lambda containing 600,000 phages was screened with labeled cDNA synthesized from an mRNA fraction enriched for the specific cell cycle gene message. Plaques containing DNA inserts which hybridized to the cDNA were picked, and their DNAs were assayed for transient complementation in DNA transformation experiments. The transient complementation assay we developed is suitable for most cell cycle genes and indeed for many genes whose products are required for cell proliferation. Of 845 phages screened, 1 contained an insert active in transient complementation of the ts cell cycle mutation. Introduction of this phage into the ts cell cycle mutant also gave rise to stable transformants which grew normally at the restrictive temperature for the ts mutant cells.

Adult↗
PubMed1986

The homeo domain of a murine protein binds 5' to its own homeo box.

Nuclear protein extracts from day 12.5 mouse embryos were used to study protein binding to DNA sequences 5' of the Hox 1.5 homeo box. Embryos of this developmental stage are known to express this gene. DNA binding protein blotting and retardation gel techniques show that murine embryonic nuclear proteins specifically bind a 753-base pair (bp) DNA fragment from the region upstream of the Hox 1.5 homeo box. A fusion protein containing the Hox 1.5 homeo domain constructed in lambda gt11 also binds the same 753-bp DNA fragment. Specific binding of the fusion protein to the upstream DNA fragment shows that the homeo box contains the sequences required for specific protein-DNA interactions, and the 753-bp fragment contains a homeo domain binding site. These results support the hypothesis that murine homeo boxes are DNA binding domains of proteins involved in the regulation of embryonic development.

Amino Acid Sequence↗
PubMed1985

Homeo box gene complex on mouse chromosome 11: molecular cloning, expression in embryogenesis, and homology to a human homeo box locus.

The homeo box is a 180 bp protein-coding domain found within homeotic genes of Drosophila and conserved in a variety of invertebrate and vertebrate species. It has been suggested that the mammalian homeo box sequences may play a role in controlling pattern formation during embryogenesis. We report findings that support this hypothesis. We have cloned three overlapping recombinant phage clones that cover a region of mouse chromosome 11 that contains a cluster of four homeo boxes (the Hox-2 locus). This locus encodes multiple transcripts that are expressed during embryogenesis. Forty kilobases of the Hox-2 region is devoid of repetitive elements and shows extensive homology with the human Hox-2 locus. These results provide direct evidence for genetic expression during embryonic development, a conserved organization in comparison to the cognate human locus, and a complexity of organization and transcript expression similar to that found in Drosophila.

Animals↗