PubMed Health⌕ Search

Biomedical subjects

L N Lukens

Publications and source records attributed to L N Lukens.

At least 19 recordsLinked to original sources
PubMed1998

Cloning and characterization of a novel sequence-specific single-stranded-DNA-binding protein.

The promoter region of the chicken alpha2(I) collagen gene contains a pyrimidine-rich element that is well conserved in different mammalian species. This sequence can also form an unusual DNA structure as shown by its sensitivity to SI nuclease in vitro and it lies in a region that is DNase I-hypersensitive only when this promoter is active. We have recently reported that fibroblast nuclear proteins, including chicken Y-box-binding protein 1, bind to this single-stranded pyrimidine-rich sequence. Here we report the isolation, from a chick embryo fibroblast cDNA expression library, of a partial cDNA clone encoding a previously unknown protein, designated SSDP (sequence-specific single-stranded DNA-binding protein), that binds this single-stranded sequence. This clone contains 1199 bp of chicken sequence and has a single long open reading frame that encodes 284 amino acid residues. The affinity-purified recombinant protein encoded by this cDNA binds sequence-specifically to the single-stranded pyrimidine sequence. This cDNA sequence lacks significant similarity to any known gene in the data banks, but it is highly conserved in expressed sequence tags derived from both mouse and human. The corresponding amino acid sequence is remarkably conserved, having 97% identity with mouse and human expressed sequences. The corresponding mRNA is approx. 1800 nt in length and is expressed in both fibroblasts and chondrocytes. The high affinity of this protein for this conserved pyrimidine-rich region suggests that it might be involved in the transcriptional regulation of the alpha2(I) collagen gene.

Amino Acid Sequence↗
PubMed1996

Y-box proteins interact with the S1 nuclease-sensitive site in the chicken alpha 2(I) collagen gene promoter.

The sequence of the chicken alpha 2(I) collagen promoter from -712 to -85, relative to exon 1, has been shown to be important for transcriptional activity. Within this region a pyrimidine/purine asymmetrical element at -200 bp forms an in vitro S1 nuclease-sensitive site. The pyrimidine-rich strand of this element interacts specifically with single-stranded DNA-binding proteins present in fibroblast nuclear extracts [Bayarsaihan and Lukens (1996) Biochem. J. 314, 293-296]. To identify these proteins we performed expression screening of a chick embryo fibroblast cDNA library using a single-stranded polypyrimidine sequence derived from this element. One of the isolated clones was found to encode a member of the cold-shock gene family, either chicken YB-1 or a highly homologous protein. This protein and a known chicken Y-box protein were both found to bind sequence-specifically to the pyrimidine-rich strand of the pyrimidine/purine asymmetrical element in the chicken alpha 2(I) collagen promoter. The binding mechanism of these proteins could be based on the formation of a non-canonical triplex DNA structure (H-DNA). Although members of this widespread and conserved protein family have been reported to modulate the expression of a number of genes, the findings reported here provide the first evidence for a possible role of cold-shock proteins in the regulation of type I collagen genes.

Animals↗
PubMed1996

Single-strand-DNA-binding factors specifically recognize the pyrimidine element in the chick alpha2(I) collagen gene promoter.

A pyrimidine element with mirror repeats centered at position -192 bp of the chick alpha2(I) collagen promoter interacts with sequence-specific DNA-binding factors. These factors bind to only the pyrimidine strand of this region and have no affinity for the complementary purine strand. Binding activity is also seen with the double-stranded form of this element, but with less affinity than to the single-stranded pyrimidine species. Southwestern blot analyses have shown that proteins of 80 and 134 kDa in chick embryo fibroblast nuclear-extracts bind to the pyrimidine strand, whereas only a 134 kDa DNA-binding protein was found in chondrocyte nuclear extracts. The binding mechanism of these nuclear proteins with single-stranded DNA might be based on a non-B-DNA conformation of the pyrimidine element. The position of this binding site in the promoter region, its potential for adopting an unusual secondary structure and the presence of the 80 kDa binding factor in chick embryo fibroblasts, but not in chondrocytes, suggest a possible role for this factor in the expression of the alpha2(I) collagen gene.

Animals↗
PubMed1995

Naturally occurring antisense transcripts are present in chick embryo chondrocytes simultaneously with the down-regulation of the alpha 1 (I) collagen gene.

It has previously been shown that very low steady state levels of alpha 1 (I) collagen mRNA are present in chick embryo sternal chondrocytes (Askew, G. R., Wang, S., and Lukens, L. N. (1991) J. Biol. Chem. 266, 16834-16841), yet nuclear run-on experiments with double-stranded cDNA probes indicated a high transcription rate at this locus. These findings were investigated in this study using single-stranded probes, where nuclear run-on experiments showed that antisense transcription of the alpha 1 (I) collagen gene was occurring in chondrocytes, while sense strand transcription was down-regulated. Treatment of these chondrocytes with 5-bromo-2'-deoxyuridine (BrdU), which causes the cells to resemble their mesenchymal precursors, resulted in an antiparallel situation, where antisense transcription was lost, and instead, sense strand transcription was acquired, suggesting that the reverse switch from sense to antisense transcription occurs during chondrogenesis. Very large (> 10 kilobases) and heterogeneous antisense transcripts of moderate stability were shown to span both ends of the gene in chondrocytes, while their absence was shown in BrdU-treated chondrocytes, chick embryo fibroblasts, and a variety of other tissues. The function of these antisense transcripts is so far unknown, but their unusual chondrocyte-specific appearance, concurrent with little or no sense strand transcription, suggests a possible functional role in the down-regulation of the alpha 1 (I) collagen gene.

Animals↗
PubMed1994

Cell-type specific promoters of the chick alpha 2(I) collagen gene in chondrocytes and fibroblasts.

Two different promoter regions responsible for the cell-type specific transcripts of the collagen alpha 2(I) gene in chick embryonic fibroblasts and chondrocytes are described. A region including part of intron 1 of the fibroblast alpha 2(I) gene is able to promote transcription in chondrocytes. This promoter region is located 5' to the chondrocyte-specific transcript, from -2205 to +42, relative to the chondrocyte transcription start site. When it is placed upstream of a luciferase reporter gene in transiently transfected chondrocytes, luciferase activity is stimulated 78-fold. The region between -2205 and -1298 is most important for the activity of the chondrocyte promoter, since a truncated promoter from -1298 through +42 gives only 15% as much activity. A similar low level of activity is given by the sequence -96 through +42. In transfected fibroblasts, a region from -1600 through +46, relative to the fibroblast transcription start site, stimulates transcription of a luciferase reporter gene by more than 1,000-fold. Deletions from -1185 through -807 and from -707 through -90 reduce promoter activity to 22% and 32%, respectively, of that given by the intact promoter. The chondrocyte and fibroblast promoters are each cell-type specific and are relatively inactive in the other cell type. Subregions within the fibroblast promoter, from -1154 through -1055, from -712 through -85, and the sequence containing the inverted CCAAT motif from -93 through -67, differentially bind factors in fibroblast but not chondrocyte nuclear extracts.

Animals↗
PubMed1991

Association of a change in chromatin structure with a tissue-specific switch in transcription start sites in the alpha 2(I) collagen gene.

Chick embryonic sternal chondrocytes do not synthesize alpha 2(I) collagen until they are shifted by treatment with 5-bromo-2'-deoxyuridine (BrdUrd) to a fibroblastic phenotype, yet they transcribe this gene as rapidly as BrdUrd-treated cells. To examine further this transcription, the DNase I hypersensitive sites were mapped in the 5' region of this gene in chondrocytes, BrdUrd-treated chondrocytes, fibroblasts and three types of non-transcribing cells. A DNase I hypersensitive site at -200 bp, previously shown to be associated with the active transcription of this gene in fibroblasts, is not present in chondrocyte chromatin. The chondrocyte alpha 2(I) gene contains, however, a novel major hypersensitive site in the DNA region corresponding to the fibroblast intron 2, near the chondrocyte-specific transcription initiation site of this gene. This novel hypersensitive site is associated with the use of this alternate start site by chondrocytes, since it is lost when BrdUrd treatment causes these chondrocytes to switch to the initiation of transcription at the fibroblast start site. The BrdUrd-treated chondrocytes contain the same alpha 2(I) hypersensitive sites as fibroblasts, except that fibroblasts have an additional, previously unreported, site at -1000 bp.

Animals↗
PubMed1991

Different levels of regulation accomplish the switch from type II to type I collagen gene expression in 5-bromo-2'-deoxyuridine-treated chondrocytes.

The shift of chick embryo chondrocytes to a fibroblastic phenotype by 5-bromo-2'-deoxyuridine (BrdUrd) has been used to examine the molecular basis of the switch from type II to type I collagen gene expression. Transcription rates of each of these three collagen genes before and after this shift were measured in nuclear run-on transcription assays with double-stranded 3'-cDNA probes specific for each of these three mRNAs. Degradation rates of each of these RNAs were calculated from the rate of decrease in the concentration of each RNA after the inhibition of synthesis with actinomycin D. The shut-off of the expression of the type II collagen gene during this shift was shown to occur at the transcriptional level, since the transcription rate of this gene decreased dramatically. The decay rate of the type II mRNA (half-life of approximately 15 h) is not significantly faster in BrdUrd-treated cells. The alpha 1(I) gene is transcribed at similar rates in untreated and shifted chondrocytes, but the steady state level of alpha 1(I) RNA in chondrocytes is only 1.5% of that in shifted cells. Although the measured degradation rate of the total alpha 1(I) RNA from untreated chondrocyte cultures is approximately the same as in shifted cells (half-life of approximately 12 h), indirect evidence suggests that this alpha 1(I) RNA is derived from a low level of fibroblast contamination of these chondrocyte cultures. The alpha 1(I) RNA synthesized by untreated chondrocytes is assumed therefore to be broken down very rapidly in the nucleus. The alpha 2(I) gene is also transcribed in untreated chondrocytes at rates similar to shifted cells but, unlike alpha 1(I) RNA, its steady state level in untreated chondrocytes is approximately 30% of its level in shifted chondrocytes. The increased level of alpha 2(I) RNA in shifted cells may be regulated in part by an increase in stability of the alpha 2(I) mRNA, which has half-lives of 5.2 and 10.4 h, respectively, in untreated and shifted chondrocytes. The alpha 2(I) RNA in the untreated chondrocytes was found to have a different 5' end from that present in the BrdUrd-shifted chondrocytes or in chick embryo fibroblasts. The presence of this altered RNA in untreated chondrocytes explains the absence of synthesis of the fibroblastic alpha 2(I) collagen polypeptide chains in these chondrocytes, despite the presence of the alpha 2(I) RNA as measured with 3' probes.

Animals↗
PubMed1988

Changes in ribosomal RNA, poly(A)+ RNA, and collagen alpha 2(I) mRNA synthesis and processing during hypertonic treatment of cultured chick embryo cells.

The effect of hypertonic conditions on RNA synthesis in cultured chick embryo cells was examined. The appearance of newly synthesized 28 S, 18 S, and 4 S and 5 S RNA into the cytoplasm was found to be decreased by hypertonic conditions. The appearance of newly synthesized poly(A)+ RNA into the cytoplasm was also found to be depressed. To examine the behavior of a specific mRNA, nuclear and cytoplasmic levels of procollagen alpha 2(I) mRNA were measured during high salt treatment. While nuclear levels of this mRNA were found to increase, those of the cytoplasm fell markedly. S1 nuclease digestion studies of an intron flanked by two exons revealed that the pro alpha 2(I) collagen nuclear RNA that accumulated under hypertonic conditions was spliced. The nuclear accumulation of mRNA appears therefore to be due to a hypertonic block of nuclear-cytoplasmic transport, and not to an inhibition of RNA splicing.

Animals↗
PubMed1985

Changes in the nuclear and cytoplasmic levels of type I and type II collagen RNAs during growth of chondrocytes in 5-bromo-2'-deoxyuridine.

Chick embryo chondrocytes were grown in cell culture in the presence of 5-bromo-2'-deoxyuridine. Under these conditions, the cells attached to the tissue culture plates and became polygonal in shape and concurrently shifted their synthesis of collagen from type II to type I subunits. The changes in the levels of the corresponding collagen RNAs in the nucleus and cytoplasm were examined during this shift by dot blot and Northern blot analyses. It was found that the expression of the cartilage collagen gene, alpha 1(II), was controlled at the transcriptional level. While the type I collagen genes may also be partially controlled at the transcriptional level, both genes appear to be transcribed in chondrocyte nuclei although chondrocytes do not synthesize type I collagen. In these chondrocytes, the transcripts of the alpha 2(I) gene are polyadenylated and are apparently exported to the cytoplasm but are not translated.

Animals↗
PubMed1983

Construction and characterization of type II collagen complementary deoxyribonucleic acid clones.

The mRNA for type II collagen was purified from embryonic chick sternum or from purified sternal chondrocytes with guanidine thiocyanate as the extractant. Double-stranded cDNAs to procollagen mRNAs from sternum were synthesized and dC-tailed. After annealing with PstI-cleaved, dG-tailed pBR322, this DNA was used to transform Escherichia coli X1776. Transformed colonies were screened by colony hybridization to type I and II collagen cDNAs. Clones that preferentially hybridized to type II cDNA were characterized further. Four such cDNA clones, pCgII-2, 3, 10 and 12, with inserts of 400, 320, 260 and 750 bp, have been identified as type II collagen cDNA clones by several criteria, including their preference for hybridizing with type II rather than type I collagen mRNAs in hybrid-selected translation experiments.

Animals↗
PubMed1981

Changes in the type II and type I collagen messenger RNA population during growth of chondrocytes in 5-bromo-2-deoxyuridine.

The collagen mRNA population of cultured chick chondrocytes was examined during a 5-bromo-2-deoxyuridine-induced shift in the type of collagen synthesized. When a cell-free protein-synthesizing system was used, it was found that an increase in the translatable mRNA for type I collagen [alpha 1 (I)]2 alpha 2 was correlated with the onset of the synthesis of this collagen type by intact cells. It was also found, both in vivo and in vitro, that a normal 2:1 ratio of type I subunits is not synthesized at early times in the type II to type I shift but that the alpha 1 (I) subunit is found in excess. It would appear, therefore, that the expression of these complementary subunits is not tightly coordinated.

Animals↗
PubMed1976

Time of occurrence of disulfide linking between procollagen chains.

The disulfide bonds that link together single collagen pro-alpha chains into trimers, prior to their secretion, were found to be synthesized after the pro-alpha chains were completed and released from polysomes and not while the pro-alpha chains were still nascent. After a 4- or 8-min incubation of cell cultures derived from embryonic chick sternum with [3H]proline, the nascent chains isolated from polysome preparations contained negligible amounts of disulfide-linked chains. After a 20-min incubation, some very large disulfide-linked material was found associated with the polysome preparation, but this material proved to be noncollagenous and also appeared to represent completed, and not nascent, chains. The presence of this proline-labeled species was confirmed in analyses of whole cell lysates, in which it is present as a mixture of monomers of approximately 290,000 molecular weight and disulfide-linked dimers. Pulse-chase experiments also gave results that were consistent with the conclusion that released single pro-alpha chains are the precursors of released disulfide-linked pro-alpha chains. Such experiments also showed that pro-alpha chains could continue to form disulfide-linked species in the presence of inhibitors of peptide chain elongation.

Animals↗
PubMed1975

Identification and purification of collagen-synthesizing polysomes with anti-collagen antibodies.

Antibodies against embryonic chick bone collagen were prepared in rabbits and were purified by affinity and ion exchange chromatography until collagen-specific and RNase-free. 125I-anti-collagen antibodies were used to locate the collagen-synthesizing polysomes of 8-day chick embryo wings and legs on sucrose gradients by measuring the polysome associated radioactivity. The 125I-anti-collagen antibodies bound predominantly to polysomes in the heavy region of sucrose gradients. These binding sites could only be saturated with homologous anti-collagen antibodies. Further evidence for the specificity of this reaction was provided by a correlation of the amount of anti-collagen antibodies bound in the heavy regions of sucrose gradients with the amount of collagen being synthesized by a particular tissue. The validity of this immunochemical method was confirmed by localizing collagen-synthesizing polysomes by an independent method which utilizes their ability to incorporate [3H]proline into collagen peptides in a cell-free system. The collagen-synthesizing polysomes are found in a single, rather broad peak in these gradients. The results of shortening the centrifugation time indicate that larger species of collagen-synthesizing polysomes are not present in these tissues. Partial purification of the collagen-synthesizing polysomes may be achieved by specifically sedimenting them after treatment with anti-collagen antibodies followed by goat anti-rabbit antibodies.

Animals↗
PubMed1971

Collagen polypeptides: normal release from polysomes in the absence of proline hydroxylation.

It is not necessary that proline be hydroxylated for the completion and release of nascent collagen chains from polysomes. Hydroxylation of collagen proline in vivo normally takes place predominantly on nascent polypeptides; however, in the presence of an inhibitor of hydroxylation, unhydroxylated chains are released. These chains may subsequently be hydroxylated when the inhibition is removed. The results clarify a controversy over the site of proline hydroxylation.

Animals↗