Cloning of alpha 2u globulin cDNA using a high efficiency technique for the cloning of trace messenger RNAs

An extremely high-efficiency technique is described for cloning double-stranded (ds) cDNAs in Escherichia coli. The method, which uses two synthetic oligonucleotide linkers rather than one, results in approx. 200--500 recombinant clones per ng of ds cDNA. This technique was used to clone a cDNA comprising 95% of the full length of the mRNA of alpha 2u globulin, a male rat liver protein, which represents approx. 1% of hepatic messenger RNA. The cloned probe was applied to study the complex hormone controls of alpha 2u globulin mRNA in male and female rats.     

Hormonal regulation of the hepatic messenger RNA levels for alpha2u globulin.

The messenger RNA rat alpha2u globulin has been identified and quantitated in a cell-free translational system derived from Krebs II ascites cells. Hepatic tissue of the mature male rats which normally produce alpha2u globulin was also found to contain a high level of alpha2u mRNA. Approximately 1.6 per cent of all poly(A) containing RNA of the adult male rat liver could be accounted for alpha2u messenger activity. Female rats do not produce alpha2u globulin and no alpha2u mRNA activity could be detected in the poly(A) containing RNA fraction obtained from the livers of these animals. However, androgen treatment to spayed female rats was found to induce the parallel appearance to both alpha2u globulin and its corresponding mRNA. Both hypophysectomy and adrenalectomy which are known to reduce the level of alpha2u globulin in the urine of male rats were found also to reduce the hepatic level of alpha2u mRNA. The results indicate that hormonal control of alpha2u globulin synthesis in rat liver is achieved primarily through regulation of its translatable mRNA level and that more than one hormone may participate in this regulation.     

Differential regulation of alpha 2u globulin gene expression in liver, lachrymal gland, and salivary gland

The alpha 2u globulins, products of a highly homologous multigene family, are synthesized in the liver and submaxillary salivary glands of the rat. Although their precise function has not been ascertained, they are of interest because of the complex developmental and hormonal regulation of their tissue levels. We now report that alpha 2u globulin is synthesized in a third tissue of the rat, the extraorbital lachrymal gland. Immunocytochemical studies indicate that the distribution of alpha 2u globulin is more homogeneous in the lachrymal gland than in the liver or submaxillary gland. In situ hybridization to alpha 2u globulin RNA reveals specific signal only over the acinar cells of the lachrymal gland. Several different isoelectric forms of alpha 2u globulin are encoded by lachrymal gland mRNA. The major lachrymal and salivary gland isoforms are indistinguishable from one another, but more acidic than the hepatic isoforms. In addition, analysis of double-stranded cDNAs with a diagnostic restriction-enzyme pair detects no differences between the alpha 2u globulin mRNAs of lachrymal and salivary gland, but clearly distinguishes these from their hepatic counterparts. In spite of the similarity between the lachrymal and salivary gland alpha 2u globulin gene products, we find that the hormonal and developmental regulation of alpha 2u globulin expression differs markedly in these two tissues. In the liver, where a different subset of alpha 2u globulin genes is expressed, a third regulatory phenotype is observed.     

The sequence of porcine αs1-casein cDNA: evidence for protein variants generated by altered RNA splicing

A cDNA library was constructed from mRNA isolated from lactating porcine mammary gland and screened with a bovine alpha s1-casein cDNA clone. Three classes of cDNA isolated varied in the number of bases within the coding region. The full length porcine alpha s1-casein cDNA is 1124bp and codes a preprotein of 206 amino acids. The other two classes of alpha s1-casein cDNA lacked 18bp and 60bp respectively when compared to the 1124-bp cDNA sequence. PCR amplification confirmed the presence of these sequences in total RNA. These differences appear to be due to altered RNA splicing.     

Cloning of the C alpha catalytic subunit of the bovine cAMP-dependent protein kinase.

The bovine C alpha type catalytic subunit of the cAMP-dependent protein kinase was cloned. A partial cDNA was isolated from a bovine heart cDNA library. This clone contained 120 bp of the coding sequence and the entire 3' untranslated region of 1431 bp. The complete coding region was cloned by PCR amplification from total bovine heart and skeletal muscle RNA. The sequence of the 3' oligonucleotide was taken from the partial cDNA clone whereas the 5' oligonucleotide was chosen by comparison of sequences of published C alpha subunits from other species. In the deduced amino acid sequence there is one deviation from the published bovine C alpha protein sequence, aspartic acid 286 is exchanged by an asparagine. The C alpha mRNA was found to be expressed differentially in various bovine tissues.     

Tissue-specific control of alpha 2u globulin gene expression: constitutive synthesis in the submaxillary gland

Synthesis of alpha 2u globulin, previously thought to occur only in the male rat liver, has now been demonstrated in the submaxillary salivary gland. Unlike liver, submaxillary synthesis of alpha 2u globulin mRNA is constitutive--that is, independent of the endocrine state, age and sex. Liver and submaxillary alpha 2u globulin mRNAs are of similar size, and their 5' ends map to the same region of the gene. Isoelectric focusing of in vitro translation products revealed that submaxillary mRNA encodes a more acidic subset of alpha 2u globulins than does liver. Salivary alpha 2u globulin mRNA manifests 5% nucleotide divergence, encoding 20 amino acid substitutions, which specifies a more acidic polypeptide than its hepatic counterpart. Thus the liver and submaxillary gland synthesize alpha 2u globulin from different sets of genes that are subject to very different developmental and hormonal control.     

Construction and characterization of a cDNA clone containing a portion of the bovine prolactin sequence.

Poly(A)-containing RNA from the bovine anterior pituitary has been used as a template for the enzymatic synthesis of double-stranded cDNA. The resulting double-stranded cDNA was inserted into the Pst I site of pBR322 with the oligo(dG)-oligo(dC) tailing technique and subsequently cloned in E. coli chi 1776. Clones containing sequences complementary to prolactin mRNA were identified by colony hybridization with partially purified prolactin cDNA. A 250 base pair sequence from one prolactin positive clone was extensively characterized and shown to contain the coding information for amino acids 119-192 of authentic bovine prolactin. The recombinant DNA from this clone was covalently attached to diazotized aminocellulose and used to purify prolactin mRNA from a mixture of mRNAs.     

Messenger RNA for alpha 2u-globulin of rat liver. Purification, partial characterization of the mRNA, and synthesis of a Hae III restriction fragment as its cDNA probe

The mRNA for the androgen-dependent hepatic protein, alpha 2u-globulin is normally present in the liver of mature male rats to the extent of about 1% of the total mRNA population. alpha 2u mRNA which was found to migrate as a 14 S band was purified about 18-fold through preparative urea-agarose gel electrophoresis. 32P-Labeled cDNA synthesized with this partially purified alpha 2u mRNA was used as substrate for two restriction endonucleases Hha I and Hae III. Digestion of the cDNA with Hha I failed to reduce its electrophoretic heterogeneity. However, Hae III digestion of the cDNA preparation greatly reduced the molecular complexity and produced several distinct cDNA bands. One of these Hae III fragments (Band A) containing 410 nucleotide residues was extracted from polyacrylamide gel and found to be complementary to alpha 2u mRNA. The identity of this cDNA fragment was established by its ability to inhibit selectively the translation of alpha 2u mRNA in the rabbit reticulocyte cell-free system and by its hybridization kinetics with poly(A)+ hepatic RNA from animals with different rates of alpha 2u synthesis. The relative R0t 1/2 values showed a direct correlation between mRNA sequences complementary to the cDNA fragment (A) and to both translatable alpha 2u mRNA and hepatic level of alpha 2u-globulin in adult male, female, and maturing male rats. Thus, the cDNA fragment containing 410 nucleotide residues generated by the restriction cleavage with Hae III can be used as a convenient probe for identification and quantitation of alpha 2u mRNA under different physiological and experimental conditions.     

Synthesis and immunocytochemical localization of alpha 2u globulin in the duct cells of the rat submaxillary gland

Alpha 2u globulin, a protein of unknown function so far believed to be synthesized exclusively in the male liver under multihormonal control, is now shown to be localized by immunocytochemistry in the granular convoluted tubules of the adult male submaxillary gland. In addition, using Northern blot analysis, we have shown specific alpha 2u globulin mRNA sequences in the RNA extracted from the submaxillary gland. Thus, it is evident that the protein is being synthesized therein. Alpha 2u globulin was also detected in the submaxillary gland duct cells of adult female and immature animals of both sexes, all of which are known not to synthesize alpha 2u globulin in their livers. The present data have established that alpha 2u globulin is synthesized in the rat submaxillary gland and indicate that the control of alpha 2u globulin gene expression in the rat liver and in the submaxillary gland is different.     

Cloning of cDNA sequences for an Artemia salina hnRNP protein: evidence for conservation through evolution.

A cDNA clone was isolated for Artemia salina protein HD40, a component of heterogenous nuclear ribonucleoproteins. Enriched Artemia 15S poly(A)+ RNA was used as a template and double-stranded cDNA sequences were inserted into the Pst I restriction endonuclease site of E. coli plasmid pBR322. Recombinant colonies were analyzed by positive hybrid selection of poly(A)+ RNA that directs the synthesis of protein HD40 in an in vitro assay. In vitro translation of the mRNA selected by recombinant clone 87HD yields a protein that is immunoprecipitated by anti-HD40 antibodies and that comigrates with authentic HD40 on gel electrophoresis. Partial proteolysis of protein HD40 and the in vitro translated product selected by clone 87HD produces the same peptide patterns. The size of the cloned insert is about 820 bp. The length of HD40 mRNA as determined by Northern blot analysis, is about 1500 nucleotides. Southern blot analysis performed with DNA of different species (plant, avian, mammal) shows cross-hybridizing bands when probed with clone 87HD DNA suggesting that the HD40 gene is evolutionarily conserved.