Supersymmetry algebra of antisymmetric tensors with Green-Schwarz mechanisms.
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Journal Article↗
Biomedical subjects
M Bodner
Publications and source records attributed to M Bodner.
Purification of growth hormone-specific transcription factor GHF-1 containing homeobox.
Pituitary-specific expression of the growth hormone (GH) gene is governed by a transcription factor, GHF-1, that binds to two sites within its promoter. Recently, GHF-1 was shown to be a member of the homeobox family of DNA-binding proteins. An important question is whether GHF-1 controls the expression of other pituitary specific genes, such as prolactin (Prl), expressed in closely related cell types. To this end, GHF-1 was purified from extracts of GH- and Prl-expressing pituitary tumor cells and identified as a 33-kilodalton polypeptide. Although GHF-1 bound to and activated the GH promoter, it did not recognize the Prl promoter. However, at least one other factor in the same extracts, which was easily separated from GHF-1, bound to several sites within the Prl but not the GH promoter. Antibodies to GHF-1 did not react with the Prl binding activity. These results imply that the pituitary-specific expression of GH and Prl is governed by two distinct trans-acting factors.
DNA-Binding Proteins↗
The pituitary-specific transcription factor GHF-1 is a homeobox-containing protein.
Growth hormone factor 1 (GHF-1) is a pituitary-specific transcription factor that plays a critical role in cell type-specific expression of the growth hormone (GH) gene. Here, we describe the isolation of bovine and rat GHF-1 cDNA clones. These cDNAs encode proteins whose molecular mass, 33K, is identical to purified GHF-1 and whose sequence agrees with a partial GHF-1 peptide sequence. The predicted GHF-1 sequence contains a region, near its C-terminus, that exhibits considerable similarity to a homeobox consensus sequence. DNAase I footprinting with bacterially expressed fusion protein containing a fragment of GHF-1 encompassing the homeobox indicates that this region of the protein functions as its DNA binding domain. Expression of GHF-1 is restricted to cells of the somatotropic lineage in the pituitary. This remarkable specificity of GHF-1 expression correlates with the selective transcription of its target, the GH gene. Other mammalian homeobox-containing proteins may function similarly as transcription factors controlling cell type-specific expression in other locations.
Amino Acid Sequence↗
A pituitary-specific trans-acting factor can stimulate transcription from the growth hormone promoter in extracts of nonexpressing cells.
The growth hormone (GH) gene is specifically expressed in the anterior pituitary. The first 289 bp of the human (h)GH 5' region contains a promoter that is active only in pituitary-derived cell lines. These cell lines also contain a specific factor, GHF-1, that binds to two sites upstream to the hGH TATA box. Using in vitro transcription systems we demonstrate greater hGH promoter activity in extracts of GH-expressing GC cells than in extracts of nonexpressing HeLa cells. The two GHF-1 binding sites are essential promoter elements in GC extracts. Addition of GHF-1-containing fractions to HeLa extracts stimulates hGH promoter activity. Thus, GHF-1 appears to be a positively acting, cell-type- and promoter-specific transcription factor. Furthermore, the tissue specificity of the hGH promoter appears to be positively controlled by the abundance or activity of GHF-1.
Animals↗
Tissue-specific expression of the human growth hormone gene is conferred in part by the binding of a specific trans-acting factor.
The molecular basis for the pituitary-specific expression of the human growth hormone (hGH) gene was investigated, by gene transfer and protein footprinting experiments. Plasmid constructs in which CAT or Neo transcription units are fused to a 0.5 kb fragment of the hGH 5' sequences were efficiently expressed in GC and GH3 cells, derived from a pituitary tumor, but not in cell lines of other origins, indicating the presence of a tissue-specific promoter. DNaseI footprinting experiments have identified at least three factors that specifically bind to the hGH 5' region. While two of these factors were also detected in extracts of non-expressing cells, the third factor, GHF-1, was detected only in extracts of GH expressing pituitary tumor cells. Mutagenesis experiments suggest that binding of GHF-1 and some of the other more ubiquitous factors is required for optimal hGH promoter activity in vivo. Tissue specificity of the hGH promoter therefore seems to be determined by the binding of at least one tissue-specific trans-acting factor, acting in concert with several other more ubiquitous, yet specific, DNA binding proteins.
Animals↗
Structure and expression of the vasoactive intestinal peptide (VIP) gene in a human tumor.
To identify the VIP biosynthetic pathways, we have isolated the human VIP gene, using synthetic oligodeoxynucleotides. These specific hybridization probes were constructed according to the neuroblastoma VIP-cDNA sequence and contained up to 39 bases. The gene structure was deduced by direct chemical nucleotide sequencing. Six exons were thus far discovered; among them two short exons, one encoding VIP and the second encoding PHM-27 (a peptide having a N-terminal histidine and C-terminal methionine amide, closely related in sequence and activity to VIP). As a model system for VIP gene expression, we used a human buccal tumor producing elevated amounts of VIP. In these cells, a major transcript of the VIP-gene was identified as a long RNA containing intron sequences. The occurrence of elevated quantities of a high molecular weight, intron containing, gene transcript which is not processed directly into mature RNA suggests that VIP gene expression may be regulated at the RNA processing level.
Base Sequence↗
Curved DNA: design, synthesis, and circularization.
Curved DNA molecules and unusually small circles have been obtained by ligation of synthetic 21-base precursors: (sequence in text). The ligation resulted in the formation of double-stranded oligo-(precursor)s possessing a strong 10.5-base-pair (bp) periodicity of the runs of adenines. Two-dimensional polyacrylamide gel electrophoresis of the ligation products showed two distinct families of spots: (i) noncircular oligo(precursor)s of 21 to 231 bp (1- to 11-mers) and (ii) four circles from 105 to 168 bp (eluted and analyzed by denaturing gel electrophoresis). The noncircular oligomers exhibited anomalously slow migration, as if they were as much as three times longer than they actually are. The amount of circular products peaked sharply at approximately equal to 126 bp, near which size the circles have been estimated to be nonconstrained both torsionally and in terms of bending. The nonconstrained circularization provides a technique for the direct measurement of the inherent curvature of DNA in solution. From the size of the circles, an estimate of 8.7 degrees is obtained for the absolute value of the AA X TT wedge angle (roll and tilt combined).
DNA↗
Coding sequences for vasoactive intestinal peptide and PHM-27 peptide are located on two adjacent exons in the human genome.
The human precursor gene for vasoactive intestinal peptide (VIP) and PHM-27, a peptide that has an NH2-terminal histidine and COOH-terminal methionine amide and is closely related in sequence and activity to VIP, was detected with synthetic oligodeoxynucleotide probes. These specific hybridization segments were constructed according to the neuroblastoma VIP cDNA sequence and contained up to 39 bases. The gene structure was partly deduced by hybridization to synthetic oligodeoxynucleotide probes and partly by direct chemical nucleotide sequencing. Four exons were discovered thus far; among them are two short exons separated by a 0.75-kilobase DNA stretch, one encoding PHM-27 and the second encoding VIP (exons 1 and 2). Each of these two exons encodes both the hormone amino acid residues as well as the post-translational processing signal sequences. The 3' splice sites of the two exons contain an identical stretch of nine nucleotides. At the cDNA level, the 3' splice sites contain the same stretch of six nucleotides, which are identically spliced. The occurrence of VIP and PHM-27 coding sequences on two separate exons of the human genome and the homology of their 3' splice site may allow alternative RNA processing as discussed below.
Amino Acid Sequence↗
Detection of mRNAs containing regulatory peptide coding sequences using synthetic oligodeoxynucleotides.
To understand the regulation of the production of peptide hormones, it is vital to elucidate their biosynthetic pathways. We chose to study a major regulatory peptide, vasoactive intestinal peptide (VIP), a peptide possessing both neurotransmitter and neurohormone actions. To identify the specific peptide mRNA we are using, as hybridization probes, radiolabeled synthetic oligodeoxynucleotides with sequence complementary to the predicted peptide mRNA sequence. Employing this approach, we identified and partially purified a approximately 1600-base long mRNA containing VIP related sequences which can be translated in vitro into VIP-immunoreactive polypeptides. Such mRNA was detected in normal VIP producing tissue (rat brain), as well as in a tumor producing VIP (human buccal tumor). This mRNA differs in size from a known VIP-mRNA identified in human neuro-blastoma cells, suggesting the possibility of different VIP-mRNAs in different cell types.
Animals↗
Keratinocyte growth factor induced epithelial proliferation facilitates retroviral-mediated gene transfer to distal lung epithelia in vivo.
BACKGROUND: Cell proliferation, vector titer and accessibility of target cells represent hurdles for efficient gene transfer to lung epithelia in vivo using recombinant murine leukemia (MuLV)-based retroviruses. We tested the hypothesis that the pulmonary epithelium is susceptible to retroviral-mediated gene transfer when stimulated to proliferate by a mitogen, keratinocyte growth factor (KGF). METHODS: Rats received keratinocyte growth factor (KGF, 2.5 micrograms/g x 4 doses, two consecutive days) intratracheally followed by high titer amphotropic retrovirus expressing beta-galactosidase. Gene transfer was assessed five days later. RESULTS: KGF stimulated transient proliferation in the bronchiolar and alveolar epithelia (30-40% PCNA positive cells at peak) which decreased to background levels seven days after administration. Gene transfer to epithelia (X-Gal positive cells) occurred more frequently in KGF treated rats, but proliferation exceeded the level of gene transfer. X-gal positive cells were noted in the alveolar epithelium and occasionally in the bronchiolar epithelium. In order to understand the discrepancy between the number of proliferating and transduced cells, primary rat tracheal epithelium cultured at the air-liquid interface was infected from either the apical or basolateral side. Gene transfer was achieved only through basolateral application of vector, suggesting that epithelial polarity represents a barrier to MuLV-based lung gene transfer in vivo. CONCLUSIONS: KGF transiently stimulates epithelial proliferation in vivo, facilitating MuLV-based gene transfer. Retroviral vectors may encounter multiple barriers which have evolved to defend the lung from infections.
Animals↗
Studies toward the biosynthesis of vasoactive intestinal peptide (VIP).
In view of the potential biological importance of VIP, we have begun to examine the regulation of its biosynthesis. For this purpose we have, as a first step, searched for an enriched source of VIP biosynthesis. By a combination of chromatographic procedures and radioimmunoassays we discovered an as yet unknown source for VIP production, namely a human buccal tumor, containing 0.67 +/- 0.05 ng VIP/micrograms protein which is greater than the richest source in brain (the cerebral cortex). Thus, we decided to use the tumor tissue for VIP-mRNA purification and characterization. To identify VIP-mRNA we are using as hybridization probes, synthetic oligodeoxynucleotides with relatively unambiguous nucleotide sequence complementary to the predicted VIP-mRNA sequence. These probes are synthesized, using the deoxynucleoside phosphoramidite approach, to a length of 17 bases each, and contain all the possible DNA sequences according to the genetic code. These specific probes are then radioactively labelled using the reaction catalyzed by the enzyme polynucleotide kinase and afterwards hybridized to mRNA, which had been resolved on denaturing agarose gels. Employing this approach, we identified a single putative VIP-mRNA band which was then partially purified by sucrose gradient centrifugation. Upon in vitro translation in a rabbit reticulocyte lysate cell free system, this mRNA was found to code for VIP immunoreactive proteins. In conclusion, our studies suggest the existence of high molecular weight precursors to VIP cross-reactive with anti-VIP antibodies, that are coded for by a partially purified mRNA containing VIP sequences.
Base Sequence↗