A seven-base-pair deletion in an intron of the albumin gene of analbuminemic rats.

Analbuminemic rats, which genetically lack serum albumin, have a mutation affecting albumin mRNA processing. Serum albumin genes were cloned from analbuminemic and normal parental Sprague-Dawley rats. Structural analyses of the two albumin genes showed that the gene from analbuminemic rats had a seven-base-pair deletion in an intron. The deletion extended from base 5 to base 11 from the 5' end of intron HI of the albumin gene. This deletion converted the sequence, G-T-A-G-G-T, which is normally located at the 5' end of intron HI, to G-T-A-G-C-G. RNA blot hybridization of analbuminemic and normal rat liver nuclear RNA using a DNA fragment containing the intron HI as a probe showed that this intron sequence persisted in albumin mRNA precursors of analbuminemic rats.     

Absence of albumin mRNA in the liver of analbuminemic rats.

Albumin synthesis in the liver of analbuminemic rats, established as a strain from a stock of Sprague-Dawley rats, was examined in vivo by labeling protein by intraperitoneal injection of L-[3H]leucine. Albumin was not synthesized in the liver of analbuminemic rats, whereas its synthesis amounted to about 14% of the total protein synthesis in the liver of normal rats. The RNA content and size distribution of the total polysomes in the liver of analbuminemic rats were not significantly different from those of normal rats. However, no functional mRNA coding for albumin was found in poly(A)-containing RNA from the liver of analbuminemic rats when tested with a cell-free translation system derived from rabbit reticulocytes. Moreover, the amount of the RNA sequence that could hybridize to purified albumin cDNA was more than 750 times greater in the liver of normal rats than in that of analbuminemic rats.     

Androgen regulation of canine prostatic arginine esterase mRNA using cloned cDNA

Canine prostatic arginine esterase complementary DNA has been cloned in pPBS27, a new cloning vector. The relative abundance of androgen-regulated mRNA in intact dog prostate was reflected by the finding that a high proportion of the clones in the cDNA library hybridized strongly by plaque or colony hybridization with a poly(A)+ RNA probe from intact dog prostate but not with a poly(A)+ RNA probe from castrated dog prostate. One clone carrying a 400 base pairs cDNA insert was selected for further studies. Translation of the hybrid-selected RNA in a cell-free system resulted in the production of a 31 kDa peptide immunoprecipitable by antibodies against arginine esterase. This identification was confirmed by partial sequence analysis of the cDNA revealing an encoding protein with high homology to known kallikreins. Northern blot analysis of poly(A)+ and total RNA showed that arginine esterase mRNA had an approximate size of 1.0 kb which corresponded to a major androgen-regulated RNA species that could be observed after denaturing agarose gel electrophoresis of prostatic poly(A)+ RNA from intact dogs. Dot-blot analysis showed that dogs which had been castrated 3 weeks before had more than 100-fold lower arginine esterase mRNA level than intact dogs or castrated dogs treated with Depo-testosterone.     

Complex population of mRNA sequences in large polyadenylylated nuclear RNA molecules.

Polyadenylylated heterogeneous nuclear RNA [poly(A)-hnRNA] from mouse brain was subjected to electrophoresis in agarose gels containing CH3HgOH, and molecules larger than 8 kilobases or 13 kilobases were recovered. cDNA was then transcribed from polyadenylylated RNA fragments cleaved from these large molecules. The resulting cDNA hybridized almost to completion with poly(A)-mRNA isolated from mouse brain polysomes. From the hybridization kinetics of this cDNA with its template RNA, it was estimated that the sequence complexity of the 3'-proximal sequences (of the same average size as mRNA) of the greater than 8 kilobase poly(A)-hnRNA was about 57,000 kilobases. The sequence complexity of poly(A)-mRNA, estimated from the template-driven hybridization kinetics of its respective cDNA, was about 110,000 kilobases. It is concluded that most, if not all, of the 3'-proximal sequences of large poly(A)-hnRNA molecules are homologous with mRNA in the mouse brain and that at least 40,000 different mRNA sequences (or portions of mRNA sequences) are represented in the 3'-proximal sequences of greater than 8 kilobase poly(A)-hnRNA.     

Colonization of albumin-producing hepatocytes derived from transplanted F344 rat bone marrow cells in the liver of congenic Nagase's analbuminemic rats

BACKGROUND/AIMS: We investigated whether bone marrow cells (BMCs) of normal rats can be transformed in albumin-producing hepatocytes in analbuminemic rat livers. METHODS: BMCs (2 x 10(7)) from F344 rats (F344) were infused via the portal vein into the livers of congenic Nagase's analbuminemic rats (F344alb) immediately after 70% hepatectomy (PH). Alternatively, F344alb were hematopoietically reconstituted with F344 BMCs by whole body irradiation and BMC transplantation before PH. The recipients were examined for albumin positive (alb +) hepatocytes and albumin mRNA in the livers as well as serum albumin levels 4 weeks later. Sry3 in situ hybridization was done for the livers of female F344alb that received male F344 BMCs. RESULTS: Livers of untreated F344alb contained a few single and double alb+hepatocytes, but these did not form clusters after PH. Clusters (>3 alb + hepatocytes) were detected in livers of the recipients which were transplanted with BMCs immediately after PH as well as the reconstituted F344alb with or without PH. Normal albumin mRNA was detected in the recipient livers, and serum albumin levels were increased. Sry3 was identified in the alb+clusters in the female recipients. CONCLUSIONS: Transplanted BMCs from normal rats can increase clusters of albumin-producing hepatocytes within the liver of analbuminemic rats.     

Molecular cloning of a cDNA sequence complementary to porphobilinogen deaminase mRNA from rat.

A cDNA clone containing sequences complementary to the mRNA coding for anemic rat spleen porphobilinogen deaminase (EC 4.3.1.8) has been isolated. A cDNA library was prepared from partially purified mRNA (1% purity). This library was then screened by colony hybridization, using a cDNA probe derived from porphobilinogen deaminase mRNA further enriched (10-20% purity) by gel electrophoresis in the presence of methylmercury hydroxide. Colonies hybridizing with the probe were analyzed by hybrid-selected translation using anemic rat spleen mRNA. Four recombinant plasmids containing porphobilinogen deaminase cDNA sequences were identified by specific immunoprecipitation of the translational product from hybrid-selected mRNA. Porphobilinogen deaminase mRNA was shown to contain 1800 bases by blot hybridization analysis. The cloned cDNA sequence consists of 1500 bases. Hybridization analysis of poly(A)+ RNA from uninduced and induced mouse erythroleukemic cells indicated that induction to erythroid differentiation by dimethyl sulfoxide results in a 10-fold increase of porphobilinogen deaminase mRNA. The rat cDNA clones hybridize to the corresponding sequences encoding human porphobilinogen deaminase. This property will be useful for isolation of human gene(s) and further characterization of the molecular lesion(s) responsible for acute intermittent porphyria.     

Distribution of rat liver albumin mRNA membrane-bound and free in polyribosomes as determined by molecular hybridization

Recently, we purified rat liver albumin mRNA and prepared albumin [(3)H]cDNA. Using albumin [(3)H]cDNA in molecular hybridization experiments, we have now determined the distribution of albumin mRNA sequences in membrane-bound and free liver polyribosomes prepared by techniques in which there is high recovery of polyribosomes without evidence of degradation. By using molecular hybridization to measure specific mRNA sequence content, numerous problems could be avoided in interpretation of results as obtained by cell-free protein synthesis or immunological methods. Under these conditions, 98% of albumin mRNA sequences in polyribosomes are found in the membrane-bound fraction.     

大鼠结缔组织生长因子部分cDNA序列的克隆及其mRNA在实验性 …

目的 克隆出大鼠结缔组织生长因子（ｃｏｎｎｅｃｔｉｖｅ ｔｉｓｓｕｅ ｇｒｏｗｔｈ ＣＴＧＦ）的部分ｃＤＮＡ序列,并观察在实验性肝经大鼠肝脏中ＣＴＧＦ ｍＲＮＡ的表达改变。方法 根据小鼠ＣＴＧＦ ｍＲＮＡ序列设计引物,用ＲＴ－ＰＣＲ从大鼠肝脏总ＲＮＡ中扩增出一段４３０ｂｐ的产物,并测定其序列,将１６只雌性Ｗｉｓｔａｒ大鼠随机为胆管堵塞组（ｎ＝８）及假手术组（ｎ＝８）,６周后取大鼠提取总ＲＮＡ,利用     

Detection and partial sequence analysis of gastrin mRNA by using an oligodeoxynucleotide probe.

We have used a specific deoxyoligonucleotide probe to detect gastrin mRNA in poly(A)-enriched RNA preparations from hog antrum. The nucleotide sequence of the oligonucleotide, d(C-T-C-C-T-C-C-A-T-C-C-A), was deduced from the unique amino acid sequence Trp-Met-Glu-Glu of gastrin. When used with hog antral RNA, the dodecanucleotide is an effective primer for the synthesis of gastrin-specific cDNA as judged by nucleotide sequence analysis of cDNA isolated by polyacrylamide gel electrophoresis. We have determined an 81-nucleotide sequence corresponding to the region of the gastrin mRNA that codes for the known amino acid sequence of the G34 progastrin intermediate species, and we have demonstrated the presence of two consecutive basic residues preceding the G34 sequence in the prohormone. Hybridization of gastrin cDNA or synthetic dodecanucleotide to hog antral RNA separated by gel electrophoresis on agarose gels in the presence of methylmercuric hydroxide indicates that the mRNA coding for gastrin is about 620 nucleotides long. These results suggest that the gastrin precursor peptide contains 110-140 amino acids. This method should be of general application for detection and characterization of mRNAs corresponding to proteins of known amino acid sequence.     

Enrichment of special Novikoff hepatoma and regenerating liver mRNA by hybridization to cDNA-cellulose

Total polysomal poly(A)+ RNA of Novikoff hepatoma and of 18-hr regenerating rat liver were compared by analysis of their in vitro translational products on two-dimensional isofocusing/sodium dodecyl sulfate gels. This technique resolved the translated proteins sufficiently to permit detection of quantitative and some qualitative differences between the two mRNA populations. Excess cDNA from regenerating liver or Novikoff hepatoma, covalently linked to cellulose, was used to adsorb the complementary mRNA sequences from Novikoff hepatoma or regenerating liver. As shown by two-dimensional gel electrophoresis, the translated products of the bound mRNA fractions contained proteins common to both tissues. Novikoff hepatoma mRNA which did not bind to regenerating liver cDNA was enriched in sequences encoding for proteins 11/5.1, 15/6.8, 40/8.2, and 65/5.1 (shown as molecular weight/pI). These polypeptides were not detectable in the translational products of regenerating liver mRNA. Regenerating liver mRNA that was not bound to Novikoff hepatoma cDNA was enriched in sequences coding for proteins 12.5/4.9, 13.5/7.4, 17/8.2, 24/5.5, and 46/6.4; these proteins were not found in the translational pattern from Novikoff hepatoma. These results show that adsorption of mRNA to solid-phase cDNA provides a valuable technique for differentiating mRNA species in related tissues and for corresponding enrichment of these specific mRNAs.     

Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene.

The alcohol dehydrogenase (ADH; alcohol: NAD+ oxidoreductase, EC 1.1.1.1) gene (Adh) of Drosophila melanogaster was isolated by utilizing a mutant strain in which the Adh locus is deleted. Adult RNA from wild-type flies was enriched in ADH sequences by gel electrophoresis and then used to prepare labeled cDNA for screening a bacteriophage lambda library of genomic Drosophila DNA. Of the clones that hybridized in the initial screen, one clone was identified that hybridized with labeled cDNA prepared from a wild-type Drosophila strain but did not hybridize with cDNA prepared from an Adh deletion strain. This clone was shown to contain ADH structural gene sequences by three criteria: in situ hybridization, in vitro translation of mRNA selected by hybridization to the cloned DNA, and comparison of the ADH protein sequence with a nucleotide sequence derived from the cloned DNA. Comparison of the restriction site maps from clones of three different wild-type Drosophila strains revealed the presence of a 200-nucleotide sequence in one strain that was absent from the other two strains. The ADH mRNA sequences were located within the cloned DNA by hybridization mapping experiments. Two intervening sequences were identified within Adh by S1 nuclease mapping experiments.     

Mechanism of induction of prolactin synthesis in GH cells

Prolactin-specific RNA (RNA(PRL)) in total nuclear RNA and in cytoplasmic poly(A)(+)RNA isolated from GH (rat pituitary) cells was selectively hybridized to immobilized cloned cDNA(PRL). Agarose gel electrophoresis of the nuclear RNA(PRL) sequences eluted from the nitrocellulose filters revealed several RNA species of approximately 25-30, 18-19, and 12-13 S. Only the 12-13 S RNA species could be detected in the cytoplasmic poly(A)(+)RNA fraction. Comparative analysis of total nuclear RNA of control and thyrotropin-releasing hormone (thyroliberin)-treated cells by the reverse Southern blot technique demonstrated increased levels of all the nuclear RNA(PRL) species in hormone-treated cells. Nuclear and cytoplasmic RNA(PRL) sequences in control and treated cells were quantitated by molecular hybridization to cloned cDNA(PRL). The 2- to 3-fold stimulation of PRL production by thyrotropin-releasing hormone-treated GH(4)C(1) cells could be correlated to the corresponding increase of nuclear RNA(PRL) sequences. The hybrid strain, which produces 1/5th the amount of PRL that the parent GH(4)C(1) does, had 1/5th the amounts of nuclear RNA(PRL) sequences. Thyrotropin-releasing hormone affected neither prolactin production nor nuclear RNA(PRL) level in 928-9b cells. RNA(PRL) sequences could not be detected either in nuclei or in cytoplasm of prolactin nonproducing F(1)BGH(1)2C(1) cells. However, prolactin production could be induced and RNA(PRL) sequences could be detected in the total nuclear RNA and in cytoplasmic poly(A)(+)RNA fraction after treatment of this GH cell substrain with 5-bromodeoxyuridine. These results demonstrate that differential basal prolactin production and its modulation by thyrotropin-releasing hormone and by 5-bromodeoxyuridine can be correlated to the altered levels of nuclear RNA(PRL) sequences in the three GH cell strains.