Detection of early changes in androgen-induced mouse renal beta-glucuronidase messenger ribonucleic acid using cloned complementary deoxyribonucleic acid

beta-Glucuronidase mRNA was purified from androgen-induced mouse kidney by immunoadsorption of polysomes to protein A-Sepharose. Cell-free translation of mRNA isolated from the protein A bound RNA followed by immunoprecipitation revealed that beta-glucuronidase mRNA represented approximately 2% of the purified mRNA fraction. This mRNA preparation was used to produce complementary DNA clones by recombination with pBR322. Clones containing sequences that were enriched during the purification procedure were selected by differential colony hybridization. These were further screened for homology with beta-glucuronidase mRNA by hybrid-selected translation. A beta-glucuronidase cDNA clone, designated pGUS7, was identified by these criteria. With this plasmid, the abundance of beta-glucuronidase mRNA in total poly(A) mRNA from androgen-induced mouse kidney was estimated to be less than 0.04%. The beta-glucuronidase cDNA plasmid hybridized to a mRNA of 2.6 kb in length, which was induced in an androgen receptor dependent fashion over a time course of 21 days. Treatment of female mice with a single dose of testosterone (10 mg) revealed that beta-glucuronidase mRNA concentration begins to increase between 12 and 24 h after hormone administration.     

Cloned complementary deoxyribonucleic acid probes for untranslated messenger ribonucleic acid components of mouse sarcoma ascites cells

Mouse sarcoma ascites cells contain several abundant mRNA species that occur to a large extent in an untranslated state. RNA preparations enriched in these species were used as starting material to construct recombinant plasmids. Cloned plasmids bearing sequences homologous to four of the untranslated mRNA species were identified by translation of hybrid-selected material. These plasmids, as well as a recombinant plasmid derived from chick alpha-actin mRNA, were used as probes for the estimation of mRNA levels in polyribosomes and in small ribonucleoprotein (RNP) particles of the ascites cells. Considerable amounts of the mRNA molecules belonging to the untranslated species were present in polyribosomes as well as in mRNPs. The actin mRNA, on the other hand, was present almost exclusively in polyribosomes. The distributions obtained by the hybridization assay resembled those estimated by translation of the same RNA preparations in cell-free systems. This indicates that the mRNA molecules of a given species engaged in translation in the cells and those present as untranslated RNP particles are equally effective in cell-free translation systems.     

Cloned complementary deoxyribonucleic acid of Drosophila cells. Relationship of genome copy number to messenger ribonucleic acid abundance

Recombinant DNA plasmids containing DNA sequence complementary to poly(adenylic acid) [(poly(A)] containing RNA from the cytoplasm of Drosophila Kc tissue culture cells were constructed. The reiteration frequency in the genome of the RNA homologous to the 20 randomly selected clones was determined by two rapid methods. Of the 20, 17 were determined to be single copy, 2 were repeated several (2-4) times, and 1 was repeated approximately 10 times. The steady-state level of mRNAs homologous to the 20 cDNAs was quantitated and varied more than 160-fold. The RNAs ranged from 0.16% to less than 0.001% of the poly(A)-containing RNA.     

Molecular hybridization between rat liver deoxyribonucleic acid and complementary ribonucleic acid

RNA (cRNA) was synthesized in vitro on a template of rat liver DNA and its hybridization with rat liver DNA was studied by using the nitrocellulose-filter method. Sonication of the DNA diminished its apparent capacity to hybridize with RNA by about 50%. This is not due to cross-linkage of DNA molecules, because it could be shown that less than 2% of the sonicated DNA was cross-linked. The effect is due instead to the small size of the sonicated DNA molecules. Below a single-stranded molecular weight of 5×10⁵ the DNA showed a progressive loss of capacity to hybridize with decrease in molecular weight. Evidence is presented suggesting that the apparently diminished capacity of the DNA to hybridize is due to loss of hybridized DNA from the membrane filters. When cRNA at concentrations of up to 25μg/ml is annealed with sonicated total DNA, an apparent hybridization saturation value is found at which about 2.5% of the DNA is hybridized with RNA. Increasing the cRNA concentration tenfold brought about the hybridization of a second component of the DNA approximately equal in amount to the first. The renaturation of rat liver DNA was studied by measuring the fall in the extinction at 260nm and two different components of renaturation were observed within the reiterated fraction of DNA. By hybridizing cRNA with different fractions of rat DNA the two components of the hybridization curve are shown to correspond to the two components of the renaturation curve. The conclusion is drawn that at a cRNA concentration of 250μg/ml most of the reiterated fraction of rat liver DNA is hybridized after annealing for 16h under standard conditions (0.30m-sodium chloride–30mm-sodium citrate at 65°C). Even with such a high cRNA concentration little or no hybridization of the slowly renaturing DNA fraction occurs. It is suggested that the most highly reiterated DNA component is poorly transcribed in vitro.     

Asymmetric template function of microbial deoxyribonucleic acids: transcription of messenger ribonucleic acid

In Bacillus subtilis and Escherichia coli, pulse-labeled ribonucleic acid (RNA) synthesized during step-down growth hybridized preferentially with the heavy (H) strand of methylated albumin-Kieselguhr-fractionated deoxyribonucleic acid (DNA). At high RNA inputs, the ratio of RNA hybridized with the H strand to that hybridized with the light (L) strand was 8.7 for B. subtilis and 2.0 for E. coli. At high DNA inputs, the H/L hybridization ratio increased by a factor of two. This change in the hybridization ratio was attributable to the fraction of the pulse-labeled RNA which is in stable RNA components. The hybridization peak of pulse-labeled RNA was specifically located in the late-eluting region of the absorbance profile of the H strand. This region was considered to represent the most actively transcribing H strand templates.     

Porcine insulin-like growth factor-I (pIGF-I): complementary deoxyribonucleic acid cloning and uterine expression of messenger ribonucleic acid encoding evolutionarily conserved IGF-I peptides

In order to facilitate studies of insulin-like growth factor-I (IGF-I) expression during the pregnancy-associated development of uterus and mammary gland in the pig model, we have isolated several cDNA clones corresponding to porcine IGF-I (pIGF-I) mRNA. Sequence analysis of two cDNA fragments (sigf. 2 and sigf. 3) revealed an open reading frame encoding in order a putative 25 amino acid (aa) hydrophobic leader peptide, the mature (processed) 70 aa pIGF-I peptide and a 35 aa carboxy-terminal extension (E) peptide. The deduced aa sequence of the pIGF-I peptide is identical to human and bovine IGF-I but differs from that of rat and mouse at three and four residues, respectively. The sequences of the amino- and carboxy-terminal IGF extension peptides are also highly conserved among these species. Northern analysis using sigf. 3 as a probe revealed multiple IGF-I mRNAs (including species of 8000, 2300, and 1200 nucleotides in length) in uteri of pregnant pigs. Highest levels of the uterine IGF-I mRNAs were found at early pregnancy, when increased levels of immunoreactive tissue IGF-I were also observed. Mammary levels of IGF-I mRNAs and protein were considerably lower than that observed for uterus at the same time period. Thus, uterine production of IGF-I appears to be especially significant during early pregnancy in the pig when uterine growth, elevated IGF-I in uterine fluids, and rapid embryonic development are observed.     

Molecular cloning of rat insulin-like growth factor I complementary deoxyribonucleic acids: differential messenger ribonucleic acid processing and regulation by growth hormone in extrahepatic tissues

Two classes of insulin-like growth factor I (IGF-I) cDNAs were isolated from an adult rat liver library using a human IGF-I cDNA probe. The two types of rat IGF-I cDNA differed by the presence or absence of a 52-base pair insert which altered the derived C-terminal amino acid sequence of the E peptide, but not the 3'-untranslated region or the sequence coding for the mature IGF-I protein. When probes derived from these cDNA clones were hybridized to Northern blots of rat mRNA, specific bands of 8.6, 2.1, and 1.0-1.4 kilobases were seen. Hybridization to poly(A)+ RNA from various tissues from GH-treated and control rats demonstrated an increase in IGF-I mRNA due to GH treatment in all tissues examined.     

Hormonal regulation of rat androgen-binding protein (ABP) messenger ribonucleic acid and homology of human testosterone-estradiol-binding globulin and ABP complementary deoxyribonucleic acids

Complementary DNA clones coding for rat androgen-binding protein (rABP) were isolated from a rat testis cDNA library constructed in the bacteriophage lambda gt11. The library was screened immunochemically, using two different antibodies against rABP. The identity of the isolated clones was confirmed by epitope selection and DNA sequence analysis. The mRNA encoding rABP could be detected in the testes of 20- and 46-day-old-rats, but not in the 10-day-old rats by hybridization with 32P-labeled rABP cDNA in a Northern blot of poly(A)+-RNA fractioned by agarose gel electrophoresis. No hybridization signal was seen with poly(A)+-RNA isolated from kidney and liver. The rABP mRNA appeared as a single species with a size of 1.65 kilobase, sufficient to encode a protein of 42,000 daltons. The concentration of rABP mRNA in the testes of 37-day-old hypophysectomized rats increased after treatment with testosterone and FSH, given alone or in combination. Sequence and hybridization analysis of cDNAs for rABP, human testosterone-estradiol-binding globulin, and human ABP demonstrates that the cDNAs for human testosterone-estradiol binding globulin and human ABP have greater sequence similarity with each other than either has with rABP.