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Biomedical subjects

N Davidson

Publications and source records attributed to N Davidson.

At least 181 records · Page 10Linked to original sources
PubMed1986

Autocrine and paracrine growth regulation of human breast cancer.

Previous work from our laboratory has demonstrated that human breast cancer (BC) cells in culture can be stimulated by physiologic concentrations of estrogen. In an effort to further understand this process, we have examined the biochemical and biological properties of proteins secreted by human BC cells in vitro. We have developed a defined medium system which simultaneously allows the collection of factors secreted by the BC cells, facilitates their purification and allows for an unequivocal assay of their effect on other BC cells. By both biochemical and radioimmunoassay procedures, MCF-7 cells secrete large quantities of IGF-I-like activity. The cells contain receptors for IGF-I and are stimulated by physiologic concentrations of IGF-I. Multiple additional peaks of growth stimulatory activity can be obtained by partial purification of conditioned media from human BC cells by sequential dialysis, acid extraction and Biogel P60 chromatography. These peaks are induced up to 200-fold by physiologic concentrations of estrogen. Several of these peaks cross-react in a radioreceptor assay with EGF and are thus candidates for transforming growth factors. Monoclonal antibodies (MCA) have been prepared which react with secreted proteins from the MCF-7 cells. One of these MCAs binds to material from MCF-7 and ZR-75-1 hormone-dependent BC cells only when these two lines are treated with estrogen but reacts with conditioned medium from several other hormone-independent cell lines in the absence of estrogen stimulation. This MCA is currently undergoing further characterization and evaluation of its biological potency. We conclude that with estrogen stimulation, hormone-dependent human BC cells secrete peptides which when partially purified can replace estrogen as a mitogen. Their role as autocrine or paracrine growth factors and their effects on surrounding nonneoplastic stroma may suggest a means of interfering with tumor proliferation.

Animals↗
PubMed1986

Involvement of a GTP-binding protein in mediation of serotonin and acetylcholine responses in Xenopus oocytes injected with rat brain messenger RNA.

Injection of poly(A)+ RNA from rat brain into Xenopus oocytes caused the appearance of Cl currents in response to serotonin (5-HT) and acetylcholine (ACh). Both neurotransmitters evoked two-component currents similar in their time course to the oocyte's endogenous cholinergic muscarinic response, which was shown in previous studies to be mediated by IP3 synthesis leading to Ca release from intracellular stores. The responses to ACh and 5-HT exhibited self- and cross-desensitization, i.e., application of either ACh or 5-HT inhibited the subsequent response to either one of the two transmitters. Intracellular injection of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) mimicked the 5-HT and ACh response, and also completely suppressed the response to the subsequent application of either ACh or 5-HT. Treatment of the oocytes with pertussis toxin (PTX) caused a 50% attenuation of ACh and 5-HT responses. In the membranes of both control and mRNA-injected oocytes, PTX catalyzed the ADP-ribosylation of a single Mr = approximately 40,000 protein. Injection of the purified beta gamma-subunits of transducin enhanced the 5-HT response. The 5-HT and GTP-gamma-S responses were inhibited by intracellular injection of the Ca2+ chelator, EGTA, as previously shown for the ACh response. These data suggest that ACh and 5-HT receptors, synthesized in the oocytes on the template of brain mRNA, act through a common pathway that involves (a) a guanine nucleotide binding protein and (b) IP3 production leading to Ca mobilization.

Acetylcholine↗
PubMed1986

Mapping transcription start points on cloned genomic DNA with T4 DNA polymerase: a precise and convenient technique.

We have developed a precise and convenient mapping technique for determining transcription start points (tsp) on cloned genomic DNA using T4 DNA polymerase. This method uses single-stranded (ss) M13 DNA and therefore is, unlike S1 and Exo VII nuclease mapping methods, independent of the restriction endonuclease sites present in the insert. Essentially the protocol involves the following steps: hybridizing an mRNA to an ss M13 vector containing an antisense genomic DNA sequence spanning the presumptive tsp (cap site); annealing a DNA primer (M13 sequencing primer) to the M13 DNA at a site on this DNA upstream from the 5' end of the mRNA on the template DNA; extending the DNA primer with T4 DNA polymerase towards the 5' end of the mRNA. Since T4 DNA polymerase will not displace the mRNA: DNA hybrid, synthesis is blocked at the first nucleotide of the mRNA molecule. The length of the extended DNA products can then be determined with single nucleotide resolution on denaturing sequencing gels in parallel with a sequencing ladder. We have used this approach to map the tsp of the mouse skeletal alpha-actin gene. The sensitivity of the method allows precise mapping of transcripts present as 0.02-0.05% of the total RNA. This method is particularly valuable for mapping the tsp of genes which are known to contain a large intron between the first and second exons. It can also be applied to map the 5' border of any given exon of a gene in an M13 vector or in other vectors that give ss DNAs.

Actins↗
PubMed1986

Activating elements in the promoter region of the chicken beta-actin gene.

We have examined the chicken cytoskeletal actin gene for promoter activity and for the presence of cis-acting gene transcription activating sequences. Plasmids were constructed with the beta-actin promoter or other fragments of the beta-actin gene adjacent to either the truncated Herpes simplex virus (HSK)-tk promoter driving the neo gene or the enhancerless simian virus 40 (SV40) promoter driving the chloramphenicol acetyltransferase (CAT) gene. The neo plasmids were tested for frequency of transformation of mammalian cells to G418 resistance. The CAT constructions were tested for CAT expression in both transient and stable expression systems. We find that the beta-actin promoter is very strong in all of the assay systems and is as strong as any promoter we have tested. Also, we find that there are sequences in the vicinity of the beta-actin promoter which act like an enhancer sequence in activating transcription from the truncated HSV-tk promoter and the enhancerless SV40 promoter. Constructs with these actin sequences augment the transformation frequency of the neo plasmids, and stimulate the level of CAT expression from the CAT plasmids after stable chromosome insertion but not during the transient expression phase.

Acetyltransferases↗
PubMed1986

Differential processing of RNA transcribed from the single-copy Drosophila myosin heavy chain gene produces four mRNAs that encode two polypeptides.

We report the sequence of genomic DNA at the 3' end of the single-copy Drosophila myosin heavy chain (MHC) gene and the structure and sequence at the 3' end of four MHC mRNAs. Two mRNAs, 7.2-kilobases (kb) and 8.0 kb in length, are expressed in all stages of development in which detectable levels of muscle-specific mRNAs accumulate. These mRNAs differ by alternate choice of two poly(A) sites within the same exon. Sequence information predicts that these two mRNAs can encode one MHC polypeptide. Two additional MHC mRNAs, 8.0 kb and 8.6 kb in length, are expressed only in late pupal and adult stages of development. These two stage-specific MHC mRNAs use the same poly(A) sites as the MHC mRNAs described above but have a different splicing pattern and thus include an additional exon. Sequence information predicts that these two stage-specific MHC mRNAs encode a second MHC polypeptide with a different COOH terminus.

Animals↗
PubMed1986

The complete sequence of the mouse skeletal alpha-actin gene reveals several conserved and inverted repeat sequences outside of the protein-coding region.

The complete nucleotide sequence of a genomic clone encoding the mouse skeletal alpha-actin gene has been determined. This single-copy gene codes for a protein identical in primary sequence to the rabbit skeletal alpha-actin. It has a large intron in the 5'-untranslated region 12 nucleotides upstream from the initiator ATG and five small introns in the coding region at codons specifying amino acids 41/42, 150, 204, 267, and 327/328. These intron positions are identical to those for the corresponding genes of chickens and rats. Similar to other skeletal alpha-actin genes, the nucleotide sequence codes for two amino acids, Met-Cys, preceding the known N-terminal Asp of the mature protein. Comparison of the nucleotide sequences of rat, mouse, chicken, and human skeletal muscle alpha-actin genes reveals conserved sequences (some not previously noted) outside of the protein-coding region. Furthermore, several inverted repeat sequences, partially within these conserved regions, have been identified. These sequences are not present in the vertebrate cytoskeletal beta-actin genes. The strong conservation of the inverted repeat sequences suggests that they may have a role in the tissue-specific expression of skeletal alpha-actin genes.

Actins↗
PubMed1986

The memory gene dunce+ encodes a remarkable set of RNAs with internal heterogeneity.

We have previously isolated the region of the Drosophila melanogaster X chromosome which contains the dunce+ gene and mapped the dunce2 mutation by recombination to a 10- to 12-kilobase (kb) interval (R. L. Davis and N. Davidson, Mol. Cell. Biol. 4:358-367, 1984). Here, we examine the expression of the dunce+ chromosomal region and identify the dunce+ gene within that region. A region of ca. 25 kb which contains the 10- to 12-kb interval to which dunce2 was mapped codes for polyadenylated RNAs of 9.6, 7.4, 7.2, 7.0, 5.4, and 4.5 kb in adult flies. These transcripts are encoded by the same DNA strand and share sequences of some exons, indicating that the transcripts arise from the same gene. Some genome probes internal to the ca. 25-kb coding region show transcript-specific hybridization, demonstrating alternate usage of exonic sequence information in the formation of the mature transcripts. The basis for this internal heterogeneity in RNAs is most likely alternative splicing. Two dunce mutants examined show aberrant RNA expression from this coding region, confirming that this region is the dunce gene. The developmental expression of these transcripts has been examined. The 5.4-kb RNA is present at all developmental stages. The 9.6-, 7.4-, 7.2-, and 7.0-kb RNAs are not expressed at detectable levels in embryos, but are detected in late embryogenesis and in later developmental stages. The 4.5-kb species is found in early embryos and adults, but not in intermediate stages. We discuss the remarkable transcript heterogeneity and expression pattern with respect to the important function this gene performs in neurobiological and other physiological processes.

Animals↗
PubMed1986

The Drosophila melanogaster actin 5C gene uses two transcription initiation sites and three polyadenylation sites to express multiple mRNA species.

At least six mRNAs are made from the Drosophila melanogaster act5C gene. We investigated the structures of these RNAs in detail and determined that they are heterogeneous at both their 5' and 3' ends. At the 5' end there were two nonhomologous leader exons which were alternately spliced to the remainder of the gene. These leader exons mapped to 1.7 and 0.7 kilobases, respectively, upstream of a common splice acceptor site which was eight base pairs 5' to the translation initiator AUG. Exon 1 is 147 bases in length, while exon 2 is 111 bases. A consensus TATA sequence was found roughly 30 base pairs upstream from exon 1, but none was found in the analogous position upstream of exon 2. The transcript length diversity arose principally from the use of three polyadenylation sites. This gave rise to RNA molecules with 3'-untranslated regions of roughly 375, 655, and 945 base pairs. With two start sites and three termination sites, this gene has the potential to produce six different transcripts. All six possible transcripts were present in whole fly mRNA. Transcripts containing the two different leader exons were found in roughly the same relative quantities through development. In contrast, the various 3' ends were differentially represented through development.

Actins↗
PubMed1985

Executive demand highest in marketing, information systems.

The overall comparison of senior executive demand shows that corporate CEOs see a greater need for senior managers during the next year, but both groups concur on the need for more senior executives in the long term. Although the CEOs agree on the increase in demand in marketing/sales and information systems/data processing during each time frame, hospital CEOs will require fewer senior executives in general management and their corporate peer call for fewer planning/corporate development professionals, reflecting the respective changes occurring in both sectors surveyed.

Data Collection↗
PubMed1985

High resolution mapping of in situ hybridized biotinylated DNA to surface-spread Drosophila polytene chromosomes.

We describe a method of mapping genes or transcripts on polytene chromosomes by transmission electron microscopy. We present several applications which illustrate that, in favorable cases, the method has a resolution of ca. 10 kg, and that high resolution mapping of hybridization sites relative to bands and puffs can be achieved. We mapped sites of transcription for poly-(A) RNA and present evidence which shows that these sites are localized in some bands and puffs, but are also found in interbands.

Animals↗
PubMed1985

Quantitative in situ gel electrophoretic assay for neomycin phosphotransferase activity in mammalian cell lysates.

A method for the assay of neomycin phosphotransferase activity in eucaryotic cell lysates is described. Total cytoplasmic proteins are fractionated in nondenaturing polyacrylamide gels and then allowed to react in situ with [gamma-32P]ATP and kanamycin. The reaction products are detected by blotting to phosphocellulose paper and autoradiography. The assay is linear with protein concentration and sensitive enough to detect expression in transient assays.

Adenosine Triphosphate↗
PubMed1985

The promoter of the long terminal repeat of feline leukemia virus is effective for expression of a mouse H-2 histocompatibility gene in mouse and human cells.

DNA-mediated gene transfer techniques have been used to study the effectiveness of a novel construction involving the feline leukemia virus long terminal repeat (FeLV LTR) for expressing the mouse H-2 Ld gene in mouse and human cells. In this construction, the transcription initiation (promoter) and termination (polyadenylation) functions of the FeLV LTR have been split by insertion of a promoterless H-2 gene between them. An S1 nuclease assay has been developed that makes it possible to measure accumulated LdRNA against a background of endogenous major histocompatibility antigen RNAs in mouse and human cells. In mouse cells, the H-2 Ld gene was expressed at approximately equal levels (measured as accumulated RNA) when driven either by its own promoter or by the FeLV LTR construction. In human cells, expression at the RNA level was highest when driven by the FeLV LTR. We conclude that the FeLV LTR construction is useful for expressing foreign genes in human cells.

Animals↗
PubMed1985

Mouse-Torpedo hybrid acetylcholine receptors: functional homology does not equal sequence homology.

The nicotinic acetylcholine (AcCho) receptor (AcChoR) is a multisubunit protein complex of stoichiometry alpha 2 beta gamma delta. The several subunits show homology with each other within a given species; in addition, homology is found between analogous subunits between species. We have used the phage SP6 RNA polymerase transcription system to produce single-species RNA in vitro for various AcChoR subunits from cDNAs. Injection of an equimolar mixture of RNA for the alpha, beta, gamma, and delta subunits of Torpedo californica AcChoR into Xenopus oocytes results in the appearance of functional receptors in the oocyte membrane. No response to AcCho is detected when the beta or gamma subunit RNA is omitted, and a small response is seen when the delta subunit RNA is omitted. Replacement of Torpedo delta subunit RNA by the mouse BC3H-1 cell line AcChoR delta subunit RNA leads to the formation of functional receptors that show a 3-4-fold greater response to AcCho than does the full Torpedo complex. No response is seen when the mouse delta RNA replaces Torpedo gamma RNA. By amino acid homology profile comparisons, the mouse delta subunit appears to be moderately but not highly similar to the Torpedo delta subunit; the apparent similarity to the Torpedo gamma subunit is only slightly less. Therefore, the features of the primary sequence that determine the functional delta character of the mouse polypeptide are not revealed by simple homology comparisons.

Amino Acid Sequence↗
PubMed1985

Developmental variations in the splicing pattern of transcripts from the Drosophila gene encoding myosin alkali light chain result in different carboxyl-terminal amino acid sequences.

The total sequence of the Drosophila melanogaster gene encoding the myosin light chain dissociated by alkali (MLC-ALK) has been determined. By sequence comparisons with an MLC-ALK cDNA clone and by S1 nuclease analyses, the pattern of introns and exons within the gene has been deduced. There are multiple polyadenylylation signals that can account for most of the observed heterogeneity in the lengths of mRNAs. In the 3' half of the gene, there are two alternative splicing patterns which result in mRNAs that translate to give proteins with two alternative 14 amino acid carboxyl-terminal sequences. There is developmental regulation of the selection of the above splicing sites. One splicing pattern produces an mRNA that translates into a protein used for both larval and adult musculature, whereas the other splicing pattern is used for the latter stage only.

Animals↗
PubMed1985

Characterization of the myosin light-chain-2 gene of Drosophila melanogaster.

Recombinant DNA clones encoding the Drosophila melanogaster homolog of the vertebrate myosin light-chain-2 (MLC-2) gene have been isolated. This single-copy gene maps to the chromosomal locus 99E. The nucleotide sequence was determined for a 3.4-kilobase genomic fragment containing the gene and for two MLC-2 cDNA clones generated from late pupal mRNA. Comparison of these sequences shows that the gene contains two introns, the positions of which are conserved in the corresponding rat sequence. Extension of a primer homologous to the mRNA reveals two start sites for transcription 12 nucleotides apart. The sequence TATA is not present ahead of the mRNA cap site. There are two major sites of poly(A) addition separated by 356 nucleotides. The protein sequence derived from translation of the cDNA sequence shows a high degree of homology with that for the DTNB myosin light chain (MLC-2) of chicken. A lower degree of sequence homology was seen in comparisons with other evolutionarily related calcium-binding proteins. RNA blots show high levels of expression of several transcripts during the developmental time stages when muscle is being produced. In vitro translation of hybrid-selected RNA produces two polypeptides which comigrate on two-dimensional gels with proteins from Drosophila actomyosin, although the cDNA sequence reveals only one 26-kilodalton primary translation product.

Amino Acid Sequence↗
PubMed1985

A survey of CEO succession strategies in the corporate and hospital worlds.

The results of a national survey of hospital and corporate CEOs reveal that the two groups hold very different views of executive succession and retirement. This first of two articles examines differences in choosing and preparing a successor, mandatory retirement ages, and CEOs' plans following retirement. A second article in the May issue of Trustee will examine comparative responses to the issue of survival as it pertains to hospitals and their CEOs and what actions are required to help preserve the survival of both.

Age Factors↗