Search PubMed⌕ Search

Biomedical subjects

D W Cleveland

Publications and source records attributed to D W Cleveland.

At least 163 records · Page 9Linked to original sources
PubMed1984

High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment.

Using a plasmid containing the bacterial chloramphenicol acetyl transferase gene, we have assayed for transient expression of DNA introduced into mouse L cells by a variety of transfection conditions. High efficiency uptake and expression of this foreign DNA have been achieved by modifying the DEAE dextran mediated transfection procedure of McCutchan and Pagano (1) to include a shock with either dimethyl sulfoxide or glycerol. Inclusion of the shock step can increase expression of the transfected gene a surprising approximately 50 fold. With plasmid constructs that do not replicate after transfection, we can readily detect CAT activity in an overnight autoradiographic exposure from less than 0.1% of an extract from a 60 mm dish of transfected cells. We have determined the amounts of DNA, the amount and time course of DEAE-dextran and dimethyl sulfoxide treatments, the effects of additional DNA, and the time after transfection which yield maximal expression. Overall, this transfection protocol using DEAE-dextran coupled to a shock treatment is simple, straightforward, and gives consistently high levels of expression of the input DNA.

Acetyltransferases↗
PubMed1984

Sequence of a highly divergent beta tubulin gene reveals regional heterogeneity in the beta tubulin polypeptide.

The nucleotide sequence of a chicken genomic DNA segment containing the chicken beta 4 tubulin gene has been determined. The predicted amino acid sequence of beta 4 is surprisingly divergent from that of the chicken beta 2 gene that encodes the dominant neural beta tubulin. beta 4 differs from beta 2 at 36 residue positions and encodes a polypeptide that is four amino acids longer, yielding a divergence of 8.9% between the two beta tubulin isotypes. While many of the amino acid substitutions are conservative, several involve significant alteration in the physiochemical properties of the residue. Furthermore, the amino acid substitution positions are not randomly located within the primary sequence but are distinctly clustered: major divergence occurs in the carboxy-terminal region beyond residue 430 and within the second protein coding exon segments of the genes. In addition, large regions of absolute sequence conservation are also present. Certain sequences within the heterogeneous regions are conserved in other species, indicating that these regions are under positive evolutionary selection pressure and are therefore probably essential for some aspect of beta-tubulin function. These findings strongly suggest that regional amino acid sequence heterogeneity may play an important role in the establishment of functionally differentiated beta tubulin polypeptides.

Amino Acid Sequence↗
PubMed1984

Programmed expression of beta-tubulin genes during development and differentiation of the chicken.

We have previously demonstrated that the chicken genome contains at least four different, functional beta-tubulin genes. By using gene specific probes we have now analyzed the relative levels of expression of the four encoded messenger RNA (mRNA) transcripts as a function of chicken development and differentiation. We have found that the RNA transcript from the beta 2 gene is present in large amounts in embryonic chick brain and is also preferentially expressed in spinal cord neurons, indicating that this transcript encodes the dominant neuronal beta-tubulin polypeptide. The beta 3 mRNA is present in overwhelming amounts in RNA from chicken testis suggesting that this gene encodes a flagellar or meiotic spindle tubulin. However, both of these genes are transcribed to varying, but lesser, degrees in a number of additional cell and tissue types indicating that they are not neuronal or testis specific, respectively. Beta 4' transcripts are present at moderate levels in all cell and tissue types examined, suggesting that this mRNA encodes a constitutive beta-tubulin polypeptide that is involved in an essential or housekeeping microtubule function. Transcripts from the beta 1 gene are a minor component of the beta-tubulin mRNA populations in all cells and tissues tested. Overall, we conclude that specific beta-tubulin mRNA species are expressed in markedly different ratios in different tissues in the chicken. Such developmental regulation may reflect the function(s) of the individual beta-tubulin polypeptides or, alternatively, may be required for precise control of tubulin gene expression in cells that utilize microtubules for divergent purposes.

Animals↗
PubMed1983

Glyceraldehyde 3-phosphate dehydrogenase protein and mRNA are both differentially expressed in adult chickens but not chick embryos.

We have determined the 679 nucleotide sequence of a cDNA clone which, by hybridization-translation experiments, corresponds to a 36K chick brain protein. Our studies provide a partial amino acid sequence for this protein, identifying it as chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Antisera raised against purified chicken GAPDH reacted with a 36K protein present in chick brain extracts and estimated to be the fourth most prevalent protein, as determined by either Coomassie Blue staining or by in vitro translation of chick brain mRNA. The amounts of GAPDH mRNA in chick brain, liver and muscle and adult chicken brain are similar, whereas the relative amount of adult chicken muscle GPDH mRNA is greatly elevated and that of adult liver lowered. The GAPDH protein levels showed a similar variation between tissues, suggesting that the levels of GAPDH protein are largely regulated by the amount of available GAPDH mRNA. The chicken GAPDH clone does not hybridize to rat mRNA, even though GAPDH is one of the most evolutionarily conserved proteins, indicating that selection pressures are heavier at the primary protein sequence level than at the nucleic acid sequence level for this gene, a situation contrasting to that of the tubulins.

Amino Acid Sequence↗
PubMed1983

Four unique genes required for beta tubulin expression in vertebrates.

We have isolated the four separate segments of chicken DNA which contain sequence homology to beta tubulin. With the exception of a fifth region of DNA which appears to contain only a 5' fragment of a beta gene, these four cloned sequences represent all of the beta tubulin encoding DNA in the chicken. Each gene is very similar in structure, containing three or four small intervening sequences clustered in the 5' portion of the coding region. Using RNAs prepared from a variety of cell lines and tissues, we have found five different mRNAs which carry beta tubulin sequences, two of which are encoded by the same gene. Three of these mRNAs are unexpectedly long (between 3500 and 4000 bases). However, these large mRNAs do give authentic beta tubulin translation products. Overall, we conclude that each of the four beta tubulin genes is a functional gene which is expressed in a specific program during differentiation. These data strongly suggest that four beta tubulins are necessary for proper microtubule function in vertebrates.

Animals↗
PubMed1983

Is apparent autoregulatory control of tubulin synthesis nontranscriptionally regulated?

Virtually all higher eucaryotic cells rapidly depress synthesis of new alpha- and beta-tubulin polypeptides in response to microtubule inhibitors that increase the pool of depolymerized subunits. This apparently autoregulatory control of tubulin synthesis is achieved through modulation of tubulin messenger RNA levels. In particular, in cells treated with the microtubule-depolymerizing drug colchicine, tubulin messenger RNAs are specifically and rapidly lost from the cell cytoplasm. A priori this loss may be the result of suppression of new tubulin RNA transcription, failure of newly synthesized tubulin RNAs to be properly processed or transported from the nucleus, or an increased rate of cytoplasmic tubulin RNA degradation. Although transcriptional regulation has been demonstrated for most cellular eucaryotic genes thus far investigated in detail, we found that the apparent rates of tubulin RNA transcription were essentially unchanged in isolated nuclei derived from colchicine treated or control cells. This finding argues that the principal control of tubulin gene expression in response to altered subunit pools is probably not achieved through a transcriptionally regulated mechanism.

Animals↗
PubMed1983

Expression of human alpha-tubulin genes: interspecies conservation of 3' untranslated regions.

To examine the sequence complexity and differential expression of human alpha-tubulin genes, we constructed cDNA libraries from two unrelated tissue types (epidermis and fetal brain). The complete sequence of a positively hybridizing alpha-tubulin clone from each library is described. Each is shown to represent an abundantly expressed gene from fetal brain and keratinocytes, respectively. Although the coding regions are extensively homologous (97%), the 3' untranslated regions are totally dissimilar. This property has been used to dissect the human alpha-tubulin multigene family into members bearing sequence relatedness in this region. Surprisingly, each of these noncoding regions shares very high (65 to 80%) interspecies homology with the 3' untranslated region of one of the two rat alpha-tubulin genes of known sequence. These unexpected homologies imply the existence of selective pressure on the 3' untranslated regions of some cytoskeletal genes which maintains sequence fidelity during the course of evolution, perhaps as a consequence of an as yet unidentified functional requirement.

Animals↗
PubMed1981

Multiple alpha and beta tubulin genes represent unlinked and dispersed gene families.

Cloned cDNA sequences specific for alpha or beta tubulin mRNAs have been used to show that the multigene families which encode either alpha or beta tubulin are unlinked and dispersed throughout the chicken genome. Fractions of chicken chromosomes partially purified by centrifugation on a sucrose gradient were digested with restriction endonucleases and electrophoresed on agarose gels. The DNA was transferred to nitrocellulose filters and hybridized to labeled probes constructed from cloned cDNA sequences specific for alpha or beta tubulin. We find alpha tubulin sequences on four different chicken chromosomes and beta tubulin sequences on at least two different chromosomes. Moreover, using chicken chromosomes further purified with a fluorescent cell sorter, we have been able unambiguously to localize alpha tubulin genes to chromosome 1 and chromosome 8 and two of the beta genes to chromosome 2.

Animals↗
PubMed1981

Nucleotide and corresponding amino acid sequences encoded by alpha and beta tubulin mRNAs.

Most of the mRNA sequences coding for alpha and beta tubulin in embryonic chick brain have been determined by sequencing of cloned cDNA copies of these mRNA copies of these mRNAs. From a 1,682-base pair cDNA sequence we have deduced the entire protein sequence for beta tubulin. For alpha tubulin, all but about 38 N-terminal amino acids have been deduced from the cDNA sequence. Although tyrosine has previously been shown to be post-translationally added to the C-terminus of alpha tubulin by a specific ligase, we conclude that the primary post-translational even must be the removal, not the addition of tyrosine because a terminal tyrosine is encoded by the mRNA.

Amino Acid Sequence↗
PubMed1981

Unpolymerized tubulin modulates the level of tubulin mRNAs.

Although numerous studies have suggested ways in which the assembly of cytoskeletal proteins can be regulated physiologically, less information has been generated on the regulation of the synthesis of these proteins. Ben-Ze'ev et al. recently suggested that the synthesis of tubulin in mouse 3T6 cells is affected by the state of assembly of microtubules. We have investigated the level at which this apparent modulation of tubulin synthesis takes place, using cloned cDNA probes for alpha- and beta-tubulin mRNAs to measure the amounts of tubulin mRNAs combined with immunoprecipitation of tubulin to monitor the rate of protein synthesis. We have found that in many, but not all, cell types tubulin synthesis decreases very rapidly in response to microtubule inhibitors that increase the monomer pool. This decline in synthesis is associated with decline in the amounts of both alpha- and beta-tubulin mRNAs. Kinetic studies of tubulin protein synthesis and RNA levels suggest that the tubulin monomer may regulate the rate of tubulin mRNA transcription. It is likely that tubulin synthesis can be shut off quickly in the cell as the result of short half-lives of the tubulin mRNAs, which may be as short as 1--2 hr. These data suggest that the cell exploits the instability of the tubulin mRNAs as a means to regulate precise levels of the monomer-tubulin pool.

Alkaloids↗
PubMed1981

Two distinct classes of keratin genes and their evolutionary significance.

Bacterial plasmids containing cDNA sequences specific for keratins were constructed from mRNA of cultured human epidermal cells. Two separate classes of cloned cDNAs were identified by positive hybrid selection: one class removed from total human epidermal mRNA a fraction that was translated into 56 and 58 kilodalton (kd) keratins, and the other class selected mRNAs that translated into a mixture of 50 kd and 46 kd keratins. When probes specific for these two keratin classes were hybridized with human DNA digested with a restriction endonuclease that does not cleave within the probe, two distinct patterns of about ten fragments each were observed. Most of the hybridizing genomic fragments corresponded to complete cDNA sequences, and it is estimated that each of the two classes is encoded by about 10 genes. When the probes were hybridized with DNA from different species, all vertebrates were found to contain discrete sequences homologous to both human keratin probes. Within each vertebrate species, the two probes always hybridized with approximately equal intensities to nonoverlapping sets of genomic sequences, suggesting a coordinate evolution between the two subfamilies of keratin genes. This finding has important functional implications for keratin filament assembly.

Animals↗
PubMed1980

A dispersed multigene family encoding tubulin in Drosophila melanogaster.

We have used cloned chicken cDNA sequences for alpha- and beta-tubulin to investigate tubulin gene organization in Drosophila melanogaster. Experiments using genomic Drosophila DNA from several sources indicate that there are at least four copies each of the alpha-tubulin gene and the beta-tubulin gene. In situ hybridization experiments show that both the alpha- and beta-tubulin multigene families have dispersed arrangements on the chromosome. Genes for alpha-tubulin have been localized at chromosomal bands 67C, 84B/C, 84D and 85E, while genes for beta-tubulin have been detected at bands 60A/B and 85D. alpha-Tubulin and beta-tubulin chicken cDNA sequences can be used to select a specific mRNA species from a complex mixture which translates in vitro into alpha- or beta-tubulin protein. RNA blot hybridization using the cloned chicken cDNA sequences as probes shows that the alpha- and beta-tubulin messages detected are clearly different in length, with the message for alpha-tubulin measuring approximately 2000 bases and the message for beta-tubulin containing approximately 1800 bases.

Animals↗