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Biomedical subjects

R Axel

Publications and source records attributed to R Axel.

At least 91 records · Page 5Linked to original sources
PubMed1983

In situ hybridization to study the origin and fate of identified neurons.

Egg-laying behavior in Aplysia is mediated by a set of peptides, including egg-laying hormone (ELH), which are released by a cluster of identified neurons, the bag cells. A family of neuropeptide genes which includes the gene encoding ELH along with two additional genes encoding the A and B peptides thought to initiate the egg-laying process has been isolated and their nucleotide sequence has been determined. In situ hybridization and immunofluorescence was used to explore the origin and distribution of the neurons that express this family of genes. The ELH genes are expressed, not only in the bag cells, but in an extensive system of neurons distributed in four of the five ganglia of the central nervous system. The genes for ELH are expressed in these cells early in the animal's life cycle. As a result, it was possible to use in situ hybridization to trace the cells expressing ELH to their site of origin. The cells originate outside the central nervous system in the ectoderm of the body wall and appear to migrate to their final locations within the central nervous system by crawling along strands of connective tissue.

Age Factors↗
PubMed1983

A structure for amplified DNA.

We have employed gene transfer to generate cell lines in which a chromosomal region consisting solely of defined DNA sequences has undergone gene amplification. We have analyzed recombinant clones from the amplified array to determine the physical structure of amplified DNA in the cell lines. The amplified DNA we have analyzed consists of a tandem array of at least 20 individual repeating units. The individual units are contiguous, and are joined to one another by homologous recombination between repeated sequences. At first approximation, all homologous recombinations are permitted such that crossing-over may occur between any two repeated sequences. Since individual units contain multiple repeated elements, the array is not a regularly repeating structure. The individual units within the array are heterogeneous, both in size and in sequence content. These observations suggest models of gene amplification which involve multiple cycles of unscheduled DNA replication at a single locus, followed by multiple recombination events which serve to link individual units to one another and ultimately to the chromosome.

Adenine Phosphoribosyltransferase↗
PubMed1983

The regulated expression of beta-globin genes introduced into mouse erythroleukemia cells.

We have introduced a hybrid mouse-human beta-globin gene as well as the intact human beta-globin gene into murine erythroleukemia (MEL) cells and have demonstrated that these genes are appropriately regulated during differentiation of the MEL cell in culture. The addition of chemical inducers to cotransformed cells results in a 5 to 50 fold increase in the level of mRNA transcribed from the exogenous globin gene. S1 nuclease and primer extension analyses demonstrate that these mRNAs initiate and terminate correctly. Nuclear transcription experiments indicate that induction of hybrid mRNA results at least in part from the increase in the rate of globin gene transcription. Furthermore, the induction appears to be specific for globin genes within an erythroid cell. These results permit the study of expression of the globin gene during erythroid differentiation and suggest that the specific induction of the globin gene is an inherent property of DNA sequences within or flanking the beta-globin genes. Moreover, the fact that the human and hybrid globin genes are both inducible in MEL cells suggests that these regulatory sequences are conserved between mouse and human cells.

Animals↗
PubMed1983

A single gene encodes multiple neuropeptides mediating a stereotyped behavior.

Egg laying in Aplysia is characterized by a stereotyped behavioral array which is mediated by several neuroactive peptides. We have sequenced two genes encoding the A and B peptides thought to initiate the egg-laying process, as well as a gene encoding egg-laying hormone (ELH) which directly mediates the behavioral array. The three genes share 90% sequence homology and are representatives of a small multigene family. Each gene encodes a protein precursor in which the active peptides are flanked by internal cleavage sites providing the potential to generate multiple small peptides. Each of the three genes consists of sequences homologous to A or B peptide as well as ELH. Although these genes share significant nucleotide homology, they have diverged such that different member genes express functionally related but nonoverlapping sets of neuroactive peptides in different tissues.

Animals↗
PubMed1982

A family of genes that codes for ELH, a neuropeptide eliciting a stereotyped pattern of behavior in Aplysia.

We describe a particularly advantageous experimental system for studying gene structure, expression and modulation in the nervous system. In the marine mollusc Aplysia, the bag cells, two discrete clusters of neurons, secrete a peptide of known behavioral function. This neuroactive peptide, egg-laying hormone (ELH), produces a characteristic and stereotypic behavioral repertoire, consisting first of a cessation of walking and inhibition of feeding, followed by head waving and egg laying. We have cloned the genes encoding ELH and characterized their organization and expression. At least five distinct genes for ELH exist within the chromosome. Sequence analysis of one recombinant clone unambiguously identifies a contiguous stretch of nucleotides that encodes the 36 amino acids of ELH. Transcription of this small multigene family results in the expression of at least five distinct RNA transcripts encoding ELH. The pattern of transcripts differs strikingly in different tissues: bag cells express three distinct mRNA species, whereas the atrial gland, a secretory reproductive gland, expresses two distinct mRNAs. Several other neuronal and nonneuronal tissues do not express ELH RNA. In vitro these mRNAs produce a series of long polypeptide precursors that must be processed to generate the active ELH peptide. This processing event is likely to generate several additional neuroactive peptides. Thus the same peptide, ELH, may be released in association with different combinations of other neuroactive peptides. The concept of combinatorial sets of neuropeptides, each bearing one overlapping peptide ELH, and each directing a differing pattern of behavior, greatly expands the information potential of a small set of genes.

Animals↗
PubMed1982

Gene amplification and gene correction in somatic cells.

We used gene transfer to identify frequent genetic rearrangements responsible for activating mutant genes in mammalian cells. We transformed an aprt- tk- cell with a plasmid containing a wild-type aprt gene and a truncated, promoterless tk gene. Transformants that integrate a single copy of this plasmid exhibit the aprt+ phenotype but remain tk-. Tk+ variants result from 20 to 50 fold amplification of the linked plasmid along with significant lengths of flanking DNA. They produce aberrant transcripts such that multiple genes are required to generate sufficient enzyme to convert the cell to the tk+ phenotype. One striking feature of the amplified aprt+ tk+ clones is the frequency (10(-4) ) at which aprt- tk+ mutants appear. These phenotypic requirements are such that the tk gene must remain amplified while all the amplified aprt genes become inactivated. The structure of the amplified DNA indicates that within aprt- cells, all amplified units bear identical mutations. These data suggest that these cells possess an efficient correction mechanism that maintains sequence homogeneity among repeated genetic elements.

Adenine Phosphoribosyltransferase↗
PubMed1982

The structure of the thymidine kinase gene promoter: nuclease hypersensitivity correlates with expression.

Accurate and quantitative transcription of the tk gene requires sequence elements that reside within 110 nucleotides of 5'-flanking DNA. We have determined the boundaries of a hypersensitive region in 5'DNA flanking the tk gene by analyzing the relative sensitivity of specific restriction sites clustered in this region. Five different restriction enzymes, recognizing over 50 sites in the promoter region of the tk gene, each show a preferential cleavage in nuclei at a restricted number of sites residing between positions -4 and -182. Thus the tk promoter sequences are contained entirely within a hypersensitive region. Analysis of this site in tk+ transformants, a tk- mutant and tk+ rerevertant indicates that expression of tk RNA is correlated with structural alterations in the tk promoter.

Animals↗
PubMed1982

Regulated expression of human growth hormone genes in mouse cells.

We have asked whether there are sequences around the human growth hormone gene that render this gene responsive to induction by glucocorticoid hormones. Recombinant clones encoding human growth hormone were introduced into the chromosome of murine fibroblasts by cotransformation. Exposure of cotransformants to glucocorticoids results in a three to five fold induction of human growth hormone mRNA and a similar induction in secreted human growth hormone protein. The DNA sequences required for induction reside within 500 nucleotides of 5'-flanking DNA. Fusion of this segment of 5'-flanking DNA to the structural gene sequences of a hormone-insensitive gene, such as thymidine kinase, now renders this gene responsive to glucocorticoid induction.

Animals↗
PubMed1982

The structure and function of a eukaryotic promoter.

Transformation, or DNA-mediated gene transfer, permits the stable introduction of new genetic information into a cell and therefore provides an opportunity to examine the expression of exogenous DNA sequences in the transformed host. A series of deletion mutants to analyze the sequences required for accurate and quantitative in vivo transcription of the herpes simplex virus (HSV) thymidine kinase (tk) gene has been constructed. Control of the efficiency of transcription as well as rough specification of the sites of initiation reside within sequences 40-110 nucleotides from the tk structural gene. The sequences responsible for induction of tk mRNA overlap this transcriptional control region. Analysis of the conformation of the integrated tk gene in the chromosome reveals that these DNA sequences are exquisitely sensitive to nuclease attack. The major heat shock gene of Drosophila and the human beta globin gene have also been introduced into murine cells to identify sequences responsible for induction of these finely regulated genes.

Animals↗
PubMed1981

Analysis of transcriptional regulatory signals of the HSV thymidine kinase gene: identification of an upstream control region.

We have identified a transcriptional control region of the herpes simplex virus tk gene. This finding results from in vivo transcription assays of specific deletion mutants constructed in vitro. A region located between 40 and 100 nucleotides upstream from the putative transcription start site of the tk gene can promote transcription by RNA polymerase form II in the complete absence of the mRNA-coding component of the gene. When the region is deleted enzymatically from a 5' direction, accurate transcriptional expression is reduced by a factor of 50. The small remaining level of accurate transcription is eliminated by deletion of sequences from 32 to 16 nucleotides upstream from the the structural gene. It appears that the sequences between 16 and 32 nucleotides upstream from the 5' terminus of the tk gene are required to specify the exact start site of transcription. Control of both the efficiency of transcription and rough specification of the position of initiation, however, depends on sequences 40--100 nucleotides upstream from the tk structural gene.

Animals↗
PubMed1981

Transforming DNA integrates into the host chromosome.

A series of rat liver cotransformed cell lines have been constructed containing from 5 to 100 copies of a variant human growth hormone gene. We have used hybridization in situ to demonstrate that most, if not all, cotransformed sequences reside in a chromosome of the host cell. In each of four cell lines examined, hybridization was restricted to a single chromosomal site with no extrachromosomal sites apparent. The site was invariant within each line; however, each line revealed a different site of integration for transforming sequences. In two of the four lines, transforming DNA resided at or near the site of gross chromosomal rearrangements, in one line near an rDNA site, and in one line in the middle of an apparently normal chromosome. Thus, insertion is not restricted to a unique chromosome or chromosomal region.

Animals↗
PubMed1981

Integration, transcription, and control of a Drosophila heat shock gene in mouse cells.

Mouse L cells were transformed with a cloned 3.6-kilobase (kb) segment of Drosophila melanogaster DNA carrying the 2.25-kb transcribed sequence for the Drosophila 70,000-dalton heat shock protein (hsp70) and 1.1 kb and 0.2 kb of 5' and 3' flanking DNA, respectively. Heat shock of one of three such transformed cell lines containing multiple copies of the intact Drosophila segment induced the abundant accumulation of transcripts of the Drosophila gene, with correct or nearly correct 5' and 3' termini. This provides evidence, in accord with earlier indications, that diverse eukaryotes, including vertebrates, have heat shock systems similar to that studied extensively in Drosophila. Our results suggest that the signals for heat shock transcription and the chromosomal sites with which they interact have been highly conserved in evolution and that the regulatory sequences controlling transcription of the gene for hsp70 lie within the 3.6-kb Drosophila segment.

Animals↗
PubMed1981

Chromosome structure and DNA sequence alterations associated with mutation of transformed genes.

We have constructed a series of tk+ cell lines by DNA-mediated gene transfer to correlate chromosomal behavior and DNA sequence alterations associated with reversion to the tk- phenotype. Tk- revertants were selected from each of four well-characterized transformed cell lines containing the viral tk gene and multiple human growth hormone genes (HGH). Tk- colonies were analyzed for the presence of tk and HGH sequences by blot hybridization to restriction endonuclease cleaved DNA. Revertants were further characterized by detailed karyotype analysis and hybridization in situ. Blot hybridization of forty tk- revertants indicates that over half of the revertants delete all of the transforming DNA from the recipient chromosome. In fifteen additional revertants, significant deletion has occurred, although transforming DNA is retained. The analysis of chromosomes by Giemsa banding together with hybridization in situ reveals that the deletion of transforming DNA is never associated with loss of an entire chromosome. Reversion to the tk- phenotype, therefore, seems to involve discrete deletions of transforming DNA without apparent chromosome loss. In this restricted set of mutants, it thus seems crucial to maintain the diploid chromosomal complement.

Animals↗