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Z D Sharp

Publications and source records attributed to Z D Sharp.

36 records · Page 2Linked to original sources
PubMed1990

Cloning of the nucleic acid-binding domain of the rat HnRNP C-type protein.

A cDNA encoding the nucleic acid-binding domain of the hnRNP C-type protein has been cloned by DNA-affinity screening of pituitary-derived expression libraries. An analysis revealed sequence identity with the human C-type cDNA and demonstrated the presence of a peptide sequence contained within the single-stranded DNA-binding protein, UP2, which was absent from the human cDNA. Structural analysis of the protein encoded by the rat cDNA demonstrated a net charge of +15 with 14.56% and 6.33% lysines and arginines, respectively, and an amino acid sequence that is consistent with an extensive helix-loop-helix-turn-helix structure.

Amino Acid Sequence↗
PubMed1990

Regulation of cell-type-specific transcription and differentiation of the pituitary.

The transcription of rat prolactin and growth hormone genes in vitro requires a pituitary transcription factor, specific to certain cell types in the pituitary, which currently appears to be the PUF-I/Pit-1/GHF-1 protein. This factor binds to cis-regulatory elements in the 5' region of both genes and exerts a positive influence on transcription initiation presumably by interacting with general transcription factors. The PUF-I/Pit-1/GHF-1 transcriptional regulatory protein probably has an important role in not only the differentiation of the pituitary lactotroph/somatotroph cell lineage; it is also expressed in the early development of the nervous system but its function there is less well documented. It appears to be one member of a family of trans-activator proteins involved in differential gene expression in several cell types.

Animals↗
PubMed1989

DNA recognition element required for PUF-I mediated cell-type-specific transcription of the rat prolactin gene.

The cell-type-specific transcription of the prolactin gene in vitro is mediated through the interaction of prolactin upstream factor I (PUF-I) with a 28 basepair region of the gene promoter (-63 to -36) which contains an 18 bp A+T-rich imperfect palindrome (-63 to -46). Base substitutions were introduced into 16 of the 18 palindromic residues by targeted saturation mutagenesis. The GH3 binding and in vitro transcription assays of the mutated promoters showed that base substitutions within the 5'-ATATTCA-3' sequence located at -52 to -46 were detrimental to PUF-I binding and its cell-type-specific transcriptional enhancement activity. Transcription assays of the mutated promoters performed with several nonpituitary-derived extracts demonstrated that a distal TATA box located from -59 to -53 promotes initiation at -27. Thus, the cell-type-specific cis-acting element required by PUF-I for DNA recognition is immediately adjacent to a general TATA sequence. Base substitutions that decreased +1 transcription and PUF-I binding concomitantly increased -27 initiation of RNA in vitro. We suggest that PUF-I binding in pituitary cells potentiates +1 transcription and represses alternative TATA box activity for initiation events occurring at -27. This is the first known report of a eukaryotic DNA binding protein that has both an activator and repressor activity for a single transcription unit.

Animals↗
PubMed1989

Dual functions of a cis-acting element within the rat prolactin gene promoter.

Within the promoter region of the rat prolactin gene lies a TA-rich imperfect palindrome. The possible functions of the 18-base-pair symmetrical sequence were investigated by using an in vitro transcription system. Prolactin templates with and without the palindrome were transcriptionally assayed in both pituitary and nonpituitary extracts. Our results indicated that the palindromic sequence has at least two functions in the regulation of prolactin transcription.

Animals↗
PubMed1989

Proto-oncogene analyses in brain tumors.

The present study determined which oncogenes (N-myc, c-myc, v-sis, or v-fos) were amplified and which messenger ribonucleic acids (mRNA's) accumulated in 10 primary human brain tumors of neuroectodermal origin. The tumors included four glioblastomas multiforme, one mixed glioma (astrocytoma grade I and ependymoma), one astrocytoma grade II, one cystic cerebellar astrocytoma, one ependymoma, one ganglioglioma, and one medulloblastoma. The relative amounts of polyadenylated (poly(A)+) RNA's homologous to these genes and their copy number were determined using the RNA and deoxyribonucleic acid blot hybridization techniques. The N-myc and v-sis probes hybridized strongly to the poly(A)+ RNA from the same recurrent glioblastoma with gene amplifications (N-myc 80 copies; v-sis three to four copies). The c-myc probe hybridized strongly to the recurrent medulloblastoma without gene amplification. The amplification or abundant accumulation of mRNA's homologous to their oncogenes may be involved in tumorigenesis or the aggressiveness of these malignant brain tumors of neuroectodermal origin and may be good molecular indicators of an extremely malignant state in these tumors.

Adolescent↗
PubMed1988

Impaired proliferation response after PDGF induction in fibroblasts from Hutchinson-Guilford Progeria syndrome.

Fibroblasts from a Hutchinson-Guilford Progeria Syndrome (HGPS) patient were compared to normal human fibroblasts to determine if differences existed in growth factor mediated cell proliferation. Cultures of progeric fibroblasts were exposed individually to platelet-derived growth factor (PDGF), epidermal growth factor (EGF), platelet poor plasma (PPP) and fetal bovine serum (FBS). Autoradiographic studies using 3H thymidine showed that progeric fibroblasts had similar labeling indices relative to controls after exposure to FBS and EGF. In contrast, progeric cells made competent with PDGF and later treated with 5% PPP had a significantly lower labeling index. This and preliminary observations on fos RNA accumulation suggests the possible existence of a genetic defect in HGPS fibroblasts.

Autoradiography↗
PubMed1988

Prolactin upstream factor I mediates cell-specific transcription.

DNA sequence-specific chromatography was used to purify prolactin upstream factor I (PUF-I) approximately 10,000- to 20,000-fold from rat GH3 cells. The purified transcription factor reconstituted enhanced pituitary-specific prolactin RNA synthesis in nonpituitary in vitro transcription assays. In vitro mutagenesis demonstrated that the capacity to stimulate prolactin gene transcription was directly correlated with PUF-I binding to an A+T-rich region located from -63 to -36 in the prolactin 5'-flanking DNA. We propose that PUF-I is a critical modulator of transcriptional activity in pituitary cells and has a central role in the stimulation of prolactin gene transcription in the mammalian pituitary lactotroph.

Animals↗
PubMed1988

Expression of three viral oncogenes (v-sis, v-myc, v-fos) in primary human brain tumors of neuroectodermal origin.

We determined which viral oncogenes (v-sis, v-myc, and v-fos) were expressed in five primary human brain tumors of neuroectodermal origin (two glioblastomas multiforme, one medulloblastoma, one cystic cerebellar astrocytoma, and one ganglioglioma) and which of these oncogenes is correlated with malignancy. Using the dot hybridization technique, we determined the relative amounts of mRNA coded by these genes using the same nitrocellulose filter. The v-myc probe showed a 4- to 12-fold greater hybridization to the mRNA from two glioblastomas and the medulloblastoma (malignant group) than the mRNA from the cystic cerebellar astrocytoma or the ganglioglioma (benign group). In contrast, RNA hybridizing to v-sis and v-fos were accumulated to a greater extent in the benign tumors. These data suggest that the amount of myc expression may be correlated with the degree of malignancy of brain tumors of neuroectodermal origin.

Brain Neoplasms↗
PubMed1987

The ribosomal DNA transcription unit of the house cricket, Acheta domesticus.

A composite map representing a single ribosomal DNA repeat unit of the house cricket, Acheta domesticus, was constructed from overlapping cloned fragments. Sites in the repeat unit for nine restriction enzymes were mapped. R-loop mapping of sequences coding for 18 S and 28 S RNA demonstrates that the 58-kb ribosomal DNA repeat unit contains a novel-sized internal transcribed spacer of 8.4 kb. The existence of this large spacer was confirmed in genomic DNA, most if not all of the genomic repeat units containing such a spacer. A 15- to 17-kb ribosomal RNA precursor transcript is synthesized as predicted on the basis of the size of the internal transcribed spacer. The 5.8 S RNA gene is localized to a 1-kb sequence immediately 5' to the 28 S gene. The coding regions examined contain no intervening sequences analogous to those described within ribosomal DNA of other eukaryotes. Only 11% of the repeat unit codes for mature ribosomal RNA, while the remainder is nontranscribed (71%) and transcribed (18%) spacer DNA.

Animals↗
PubMed1987

Reconstitution of cell-type-specific transcription of the rat prolactin gene in vitro.

We present evidence for the existence of prolactin upstream factor 1 (PUF-1) in rat pituitary-derived cells and demonstrate its interaction with a symmetrical DNA element located in the 5' flanking region of the gene. An in vitro expression system developed from pituitary-derived GH3 cells was used to determine that 420 base pairs (bp) of 5' flanking DNA was sufficient for cell-specific, accurate, and efficient RNA polymerase II transcription of the rat prolactin gene. Reconstitution of in vitro transcription with pituitary and nonpituitary nuclear extracts suggested that the presence of GH3 cell-specific factors mediated the activation of prolactin gene expression. We also demonstrated that a functionally stable transcription complex assembled on the prolactin promoter. Using DNase I protection procedures, we have identified the DNA-protein binding area in the prolactin 5' flanking region. GH3 nuclear extracts contain a cell-specific protein (PUF-I) that binds to a 28-bp region (-63 to -36)which contains an 18-bp imperfect palindrome (-63 to -46). The role that the interaction between PUF-I and the imperfect palindrome plays in in vitro pituitary-specific prolactin gene expression is discussed.

Animals↗
PubMed1987

Correlation of rat pituitary prolactin messenger ribonucleic acid and hormone content with serum levels during the estrogen-induced surge.

The model of the serum PRL surge generated in the ovariectomized rat after estradiol benzoate (EB) treatment was used to study the relationship between serum and pituitary PRL levels and pituitary PRL mRNA levels. Adult ovariectomized rats were injected sc with 7 micrograms EB or vehicle at noon on day 0. Three days later (day 3), the rats were decapitated every 4 h over a 24-h period (0800 h on day 3 to 0400 h on day 4) for determination of serum and pituitary PRL and GH levels by RIA. In addition, PRL and GH mRNA content was determined using dot blot hybridization with cDNAs. Administration of EB resulted in a significant rise in serum PRL levels at 1200, 1600, and 2000 h on day 3 compared to control values. At other times, serum PRL levels in the EB group were the same as control values. EB treatment also elicited a marked increase in pituitary PRL content at all time periods examined except during (1600 and 2000 h) and after the PRL surge (2400 h on day 3) when there was a significant reduction in stored pituitary PRL. The pituitary PRL mRNA content in the EB-treated group was significantly elevated (4- to 6-fold) over control levels throughout the study. Furthermore, PRL mRNA levels in EB-treated rats were significantly higher at 2000 and 2400 h on day 3 than at other time periods. In contrast to its effects on PRL, EB treatment had a slight inhibitory effect on pituitary GH content at 2000 and 2400 h on day 3 compared to control values; otherwise, this steroid had no effect on serum GH levels and pituitary GH mRNA content. Interestingly, serum GH levels and pituitary GH mRNA content in both treatment and control groups fluctuated in a pattern consistent with circadian rhythms, with peak values occurring during the lights-on hours. These data show that estrogen has a stimulatory effect on pituitary content of PRL and its corresponding mRNA in the rat 3 days after injection. These elevated PRL mRNa levels may be necessary for the occurrence of PRL surges. Furthermore, the facts that serum PRL levels were elevated only at certain times (1200-2000 h on day 3) while PRL mRNA content was increased at all times in the EB-treated rats suggest a differential regulation between PRL release and biosynthesis.

Animals↗
PubMed1986

Analysis of a large nontranscribed spacer in the ribosomal DNA of the house cricket, Acheta domesticus (Orthoptera:Gryllidae).

An analysis of a 29-kilobase nontranscribed spacer fragment in the ribosomal DNA (rDNA) of the house cricket, Acheta domesticus, revealed a highly repetitious structure. A total of eight EcoRI repeats of three different size classes measuring 259, 420, and 508 base pairs (bp) was mapped to a region 2 kilobases (kb) from the 18 S coding region. The repeats were oriented in a nonrandom manner and had sequences homologous to DNA located immediately adjacent to the repetitive array. DNA sequence analysis showed that the repetitive region was composed of smaller direct repeats 66, 67, and 383 bp in length. There was minor length heterogeneity of the chromosomal restriction fragments containing the entire array, indicating that a variable number of EcoRI repeats is a minor contributor to the total repeat-unit length heterogeneity. Immediately upstream from the EcoRI array there is a 17-kb region composed of 50 to 60 subrepeat elements recognized by a variety of restriction endonucleases. A subcloned SmaI repeat from the array was not homologous to any other part of the rDNA repeat unit or other chromosomal DNA. There was little length heterogeneity in restriction fragments containing the chromosomal 17-kb repetitions region. Immediately upstream from the 17-Kb region there is a 4.1-kb segment with sequences homologous to the EcoRI repeats.

Animals↗
PubMed1985

Preferential DNase I sensitivity of insert-free ribosomal RNA repeats of Drosophila melanogaster.

The five predominant types of rDNA repeats in D. melanogaster were analyzed with respect to their DNase I sensitivity. Only the insert-free repeats showed a generalized DNase I sensitivity pattern whereas the major type I, both minor type I and type II repeats were not as extensively degraded by the nuclease. For XX and XY embryonic nuclei, where there is rapid cell division, the majority of the In- repeats were DNase I sensitive. This indicated that these In- repeats have the potential to be transcribed during this developmental stage. When compared to the In- repeats, the chromatin configuration of the In+ repeats is indicative of a higher order of chromatin folding. The paucity of In+ primary gene transcripts observed in vivo could result from In+ repeats being packaged into a more condensed form of chromatin.

Animals↗
PubMed1985

Vasoactive intestinal peptide increases prolactin messenger ribonucleic acid content in GH3 cells.

The influence of vasoactive intestinal peptide (VIP), TRH, and dexamethasone (Dex) on PRL mRNA was investigated in PRL-producing GH3 cells using cytoplasmic dot hybridization and RNA blot analysis. Total cytoplasmic RNA was transferred to nitrocellulose filter paper and quantitated by hybridization to PRL recombinant DNA probes labeled with 32P. Incubation of GH3 cells with VIP for 25 h increased the content of cytoplasmic PRL mRNA. This increase was dose dependent, being significant at 2 X 10(-8) M and reaching a maximum at 2 X 10(-7) M. VIP at 2 X 10(-9) M had no effect on cytoplasmic PRL mRNA content. TRH (2 X 10(-7) M) also increased whereas Dex (2 X 10(-7) M) decreased the content of PRL mRNA. The inhibitory effect of Dex (2 X 10(-7) M) on cytoplasmic PRL mRNA was reversed by VIP (2 X 10(-7) M). Changes in medium PRL levels after these various hormone treatments paralleled those changes observed in PRL mRNA content. Examination of total poly(A)+ RNA demonstrated that incubation with VIP (2 X 10(-7) M) for 6 h increased the content of the mature PRL mRNA and its processing intermediates. Dex (2 X 10(-7) M) decreased the content of all species of PRL mRNA. These data suggest that VIP-stimulated PRL release is the result of an increase in the content of PRL mRNA and its precursors.

Animals↗
PubMed1985

Estrogen induced prolactin mRNA accumulation in adult male rat pituitary as revealed by in situ hybridization.

Adult male rats were injected with different doses (1, 10, and 100 micrograms) of 17 beta-estradiol daily for 5 days, and the changes in prolactin (PRL) mRNA levels were examined by in situ hybridization and cytoplasmic dot blot hybridization using cloned cDNA for rat prolactin mRNA. An increase in cytoplasmic PRL mRNA content was evident in all the animals treated with estrogen as revealed with cytoplasmic dot blot analysis. There were however, no significant differences in PRL mRNA content among the three estradiol treated groups. Cytoplasmic PRL mRNA was also demonstrated by in situ hybridization on the frozen pituitary sections using a 3H-labeled PRL cDNA probe. The number of grains per cell was increased after estrogen treatment. 3H-thymidine uptake into pituitary cells was also examined in vivo using combined techniques of immunocytochemistry and autoradiography. Although the percentage of immunoreactive PRL cells which took up thymidine in their nuclei increased to more than double after estrogen treatment, the increase in the total number of immunoreactive PRL cells was small. These results suggest that the major effect of estrogen on PRL cells is an increase in the accumulation of PRL mRNA in the individual PRL cells. The number of grains per cell was found to vary from cell to cell, both in control and estrogen treated animals. This variability is discussed in relation to the functional heterogeneity within the PRL cell population.

Animals↗
PubMed1983

X chromosome nucleolus organizer mutants which alter major type I repeat multiplicity in Drosophila melanogaster.

The nucleolus organizer (NO) of the D. melanogaster X chromosome is composed of ribosomal repeat units which contain two types (I and II) of non-rDNA insertions (In+) and repeats with no insertions (In-). Evidence from other laboratories indicate random interspersion of all types of repeat units within the X NO. An EcoRI and BamHI examination of rDNA from two bobbed mutants, bb2rI and mal12 demonstrates segregation of the major type I repeat units. The 46 rDNA repeats of the bb2rI NO contain no detectable major type I repeats whereas the majority of the 68 rDNA mal12 repeats are major type I and tandemly linked. This observation suggests that gross deletions of rDNA can result in nucleolus organizer regions with predominantly one type of repeat unit. Additivity tests demonstrate that the 46 ribosomal repeats of the bb2rI chromosome revert the phenotype of other bobbed NOs, but the 68 mal12 ribosomal repeats show no or slight additivity. This is in agreement with the observation that In+ repeats do not significantly contribute to functional rRNA. A Southern blot analysis using BamHI which cuts only in type I insertions demonstrates that the majority of major type I In+ repeating units exist in tandem linkage group(s) within the X NO.

Animals↗
PubMed1983

A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast.

Polyethylene glycol (PEG) can induce genetic transformation in both bacteria (Escherichia coli) and yeast (Saccharomyces cerevisiae) without cell wall removal. PEG-mediated transformation of E. coli is technically simple and yields transformants with an efficiency of 10(6)-10(7) transformants/microgram DNA. Detailed analysis of the parameters involved in PEG-mediated transformation of E. coli reveals basic differences between the PEG and standard CaCl2 methods for transformation of E. coli. PEG-mediated transformation of yeast is far simpler than existing protoplast methods and is comparable in efficiency. The new methods described here for PEG-mediated genetic transformation may prove to be of general utility in performing genetic transformation in a wide variety of organisms.

Cations↗