The organization of the X-linked gene for human hypoxanthine phosphoribosyltransferase (HPRT, EC 2.4.2.8.) has been determined by a combination of restriction endonuclease mapping, heteroduplex analysis and DNA sequence analysis of overlapping genomic clones. The entire gene is 42 kilobases in length and split into 9 exons. The sizes of the 7 internal exons and the exon-intron boundaries are identical to those of mouse HPRT gene. The 5' end of the gene lacks the prototypical 5' transcriptional regulatory sequence elements but contains extremely GC-rich sequences and five GC hexanucleotide motifs (5'-GGCGGG-3'). These structural features are very similar to those found in the mouse HPRT gene and to some of the regulatory signals common to a class of constitutively expressed "housekeeping" genes. Several transcriptional start sites have been identified by nuclease protection studies. Extensive sequence homology between the mouse and human genes is found in the 3' non-coding portion of the gene.

A chimeric plasmid containing cDNA complementary to rat calcitonin mRNA has been constructed. Partial sequence analysis shows that the insert contains a nucleotide sequence encoding the complete amino acid sequence of calcitonin. Two basic amino acids precede and three basic amino acids follow the hormone sequence, suggesting that calcitonin is generated by the proteolytic cleavage of a larger precursor in a manner analogous to that of other small polypeptide hormones. The COOH-terminal proline, known to be amidated in the secreted hormone, is followed by a glycine in the precursor. (20, 21). The RNA was centrifuged through 15-30% linear sucrose density gradients containing 10 mM Tris-HCl (pH 7.4), 20 mM NaCl, and 0.1% NaDodSO4. The RNA fraction centered at 11.5 S was selected and reextracted with phenol/chloroform, 1:1 (vol/vol). After serial ethanol precipitations, this poly(A)-rich mRNA was used as template for cDNA synthesis with reverse transcriptase as described (22). The cDNA was subsequently centrifuged through 5-20% linear sucrose density gradients containing 0.1 M NaOH and 0.9 M NaCl. The region of the gradient containing DNA 800-1500 nucleotides long was made double stranded with E. coli DNA polymerase I (23). The reaction mixtures were adjusted to 0.1 % NaDodSO4 and 25 mM EDTA, extracted with phenol/chloroform, and passed over a Sephadex G-200 column. The excluded portion was reprecipitated and subjected to nuclease S1 digestion in order to break the 5' hairpin loop (23). The double-stranded DNA was extracted with phenol/chloroform and again purified by Sephadex G-200 column chromatography.Homopolymeric extension of the 3' termini of the DNA was accomplished with terminal deoxynucleotidyltransferase and dATP (24). Plasmid pBR322 DNA was cleaved with Pst I (23, 25) and homopolymeric extension was performed with dTTP.[3H]dATP and [3H]dTTP served as tracers to monitor the extent of reactions. Hybridization was carried out as described (26) except that samples were initially heated to 65°C for 10 min, incubated at 41°C for 2 hr, and allowed to cool slowly at 250C.These chimeric plasmids were used to transform E. coli K-12 strain SF8 as described (26) in accord with the biological and physical containment procedures approved under the National Institutes of Health Guidelines for Recombinant DNA Research. Transformants found to be tetracycline resistant were tested for ampicillin sensitivity. Bacterial clones containing cDNA inserts were identified by an in situ colony hybridization assay (27). Plasmids from selected colonies were amplified with 125 ug of chloramphenicol per ml and purified by the cleared lysate technique (28). Plasmid C-I was selected for sequence analysis and use in the subsequent study on the basis of restriction digests and ability to arrest the translation of calcitonin precursor mRNA by hybridization (29).

The superhelical, closed circular form of polyoma deoxyribonucleic acid (DNA) (Co 1) is bound in a 25S DNA-protein complex to the viral histone-like proteins after alkaline disruption of the virion. Nicked viral DNA or linear DNA are largely free of protein. Most of the viral protein disruption is in the form of capsomeres, sedimenting principally at 105 and 7S. Despite the relatively constant ratio of 10S to 7S material in many preparations, (1:5.5 to 1:6.0, respectively), the two classes of capsomeres are indistinguishable by electron microscopy and contain only P2, P3, and P4 in molar ratios of approximately 5: 1:1 or 6:1: 1, respectively. Material with sedimentation rates of approximately 1 to 3S is enriched for P1 and contains small amounts of P2, P3, and P4. During the in vitro reassembly of DNA-free, shell-like particles from disrupted virus, proteins P1, P2, P3, P4 , and P7 are reincorporated efficiently, whereas P5 and P6 are not. The presence in empty reassembled particles of histone-like protein, expecially P7, implies that at least this one of the minor protein components of the virion may participate in protein-protein interactions with other components of the capsid.

The tripeptide thyroliberin (thyrotrop in-releasing hormone, TRH) regulates the levels of cytoplasmic prolactin and growth hormone (somatotropin) mRNAs in a line of functional rat pituitary tumor cells. Intracellular mRNA levels were determined with specific complementary DNA probes in RNA-excess hybridization reactions and independently by two-dimensional gel electrophoretic analysis of the products of mRNA-directed cell-free translation. Prolactin mRNA sequences increased from 1.1 to 4.5% of the cytoplasmic poly(Acontaining mRNA within 48 hr after addition of TRH to the medium; in parallel, growth hormone mRNA sequences were decreased. Similar effects on prolactin mRNA levels were observed in cells grown in serum-containing medium or when the cells were maintained in a serum-free medium for 72 hr prior to addition of TRH, suggesting that TRH is responsible for the stimulation of prolactin mRNA levels rather than for exerting a permissive effect for another factor present in serum.The tripeptide pGlu-His-ProNH2, initially described on the basis of its ability to stimulate the release of thyrotropin (1, 2) and referred to as thyroliberin (thyrotropin-releasing hormone, TRH), was subsequently reported to stimulate the secretion and the apparent synthesis of prolactin 3-to 10-fold in a line of functional rat pituitary tumor cells (GH13) that synthesizes somatotropin (growth hormone) and prolactin (3,4). This stimulation of prolactin production was suggested to reflect an increase in its de novo biosynthesis; TRH did not produce an apparent alteration in the rate of prolactin catabolism (5). Addition of TRH also decreased the apparent rate of growth hormone biosynthesis (4, 5). The synthesis of growth hormone and prolactin was subsequently reported to be regulated by multiple hormones. Estrogen was also demonstrated to exert a stimulatory effect on prolactin biosynthesis (6). The addition of cortisol and/or thyroid hormone stimulated growth hormone biosynthesis 3-to 10-fold, with a concomitant decrease in the rate of prolactin biosynthesis (7,8). The rate of growth hormone synthesis was reported to decrease to very low or undetectable levels when the cells were maintained in serum-free medium; growth hormone synthesis could be stimulated by addition of thyroid hormone alone or in combination with cortisol (9).Previous studies (10-14) have shown, by cell-free translation of mRNA from cells, that the increased synthetic levels of these hormones were reflected in an increased mRNA-directed synthesis of the hormone precursors, preprolactin and pregrowth hormone (10,(15)(16)(17). In this report we compare mRNA levels quantitated by translation and two-dimensional gel analysis with mRNA levels quantitated by hybridization to cDNA probes specific for prolactin and growth hormone mRNAs. TRH increases prolactin mRNA levels while simultaneously decreasing growth hormone mRNA levels. In addition, the cell-free translation products, preprolactin and pregrowth hormone, exhibit molecular heterogeneity. (Sigma), bovine ...

Subribosomal and polyribosomal messenger ribonucleoproteins (mRNPs) were isolated from Ehrlich ascites tumor cells by a method involving sedimentation of polyribosomal and subribosomal particles, dissociation with EDTA, and rate-zonal sedimentation. The fractions containing mRNA protein particles were applied to glass fiber filters and extensively washed with buffer containing 0.5 M KCl. The eluted material was demonstrated to be an RNA-protein complex containing poly(A)-rich RNA, heterogeneous in size, and free of 18S or 28S rRNA. mRNA function for the RNA was suggested by its ability to direct protein synthesis in a cell-free protein-synthesizing system derived from wheat germ embryos. Analysis of the proteins associated with subribosomal and polyribosomal mRNPs by iodination and sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed at least seven similar proteins. The apparent molecular weights of the three most prominent proteins were 78,000, 52,000, and 34,000. Analysis of reticulocyte polyribosomal mRNPs revealed an increased prominence of the 78,000 and 52,000 molecular weight proteins relative to the other protein bands.