Changes in expression of albumin and alpha-fetoprotein genes during rat liver development and neoplasia.

Albumin mRNA was isolated and purified from rat liver polysomes by a combination of immunoprecipitation of specific polysomes, poly(U)-Sepharose 4B chromatography, and fractionation of the resulting poly(A)-containing RNA on a sucrose gradient. alpha-Fetoprotein (AFP) mRNA was isolated from Morris hepatoma 7777 by a similar procedure. The purity of the mRNA preparations was determined by analytical gel electrophoresis under denaturing conditions, analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polypeptides synthesized in a wheat germ cell-free system, and the kinetics of hybridization to cDNA transcribed from albumin mRNA and AFP mRNA. The albumin mRNA possessed a chain length of approximately 2265 nucleotides and the AFP mRNA possesed a length of approximately 2235 nucleotides when examined under stringent denaturing conditions on agarose gels containing 10 mM methylmercury hydroxide. Analysis of poly(A) content by a hybridization assay with [3H]poly(U) revealed the presence in albumin mRNA of a poly(A) region containing approximately 100 adenosine residues. The AFP mRNA preparation was found to contain an average poly(A) tract of approximately 190 bases. Thus, albumin mRNA appears to contain approximately 330 untranslated nucleotides, and AFP mRNA appears to contain a similar number (approximately 285) of noncoding, nonpoly(A) bases. The purified albumin and AFP mRNA's were used as templates for synthesis of full-length cDNA hybridization probes. Both of the probes selectively hybridized to their templates with kinetics expected for single RNA species the sizes of albumin and AFP mRNA. ROt analysis was used to quantitate albumin and AFP mRNA sequences during normal liver postnatal development and liver oncogenesis. The number of polysomal AFP mRNA molecules per liver was found to drastically decrease during the first weeks of postnatal life, concomitant with a decline in the AFP synthetic capacity of the livers and in the serum concentrations of AFP. During this period, the concentration of albumin mRNA molecules per cell in the liver remained at high, approximately constant levels. In Morris hepatoma 7777, the concentration of AFP-specifying sequences was at least 10(3)-fold higher than that found in normal adult liver, whereas the content of albumin nRNA was four- to five-fold lower. These changes in concentration of albumin and AFP mRNA sequences closely correlated with a parallel variation in the specific protein synthetic capacity of the tissues.     

Selective detection of rat and mouse specific albumin and alpha-fetoprotein mRNA molecules under highly stringent hybridization conditions

The molecular analysis of some important interactions observed between the parental genomes in interspecific cell hybrids requires the availability of highly specific hybridization assays to selectively quantitate mRNA sequences coding for the same protein but transcribed from the two different genomes. Specific hybridization techniques which should permit the selective detection of rat and mouse albumin and alpha-fetoprotein (AFP) mRNA molecules in a mixture of the two types of mRNAs are presented here. The high degree of homology existing between the AFP mRNA sequences coding for mouse and rat AFP, and, presumably, albumin, results in extensive cross-hybridization with the cDNA probes under standard hybridization conditions. No size differences could be detected between the two types of mRNA molecules from the two species. A Tm difference of 7 degrees C between the intra- and interspecific mRNA:rat cDNA hybrids allowed the establishment of highly stringent solution hybridization conditions necessary to measure separately the contents of rat albumin and AFP mRNAs. Mouse albumin and AFP cDNA clones were then isolated from mouse liver and yolk sac cDNA libraries, and used to show the usefulness of highly stringent washing conditions to discriminate between rat and mouse albumin and AFP mRNA molecules in conventional "Northern blotting" techniques. In combination with the solution hybridization assay, these filter hybridization techniques can be used to specifically quantitate the content of rat and mouse albumin and AFP mRNA molecules in interspecific cell hybrids.     

Quantitation of casein messenger ribonucleic acid sequences using a specific complementary DNA hybridization probe.

Two highly purified rat casein mRNA fractions were used as templates to synthesize complementary DNA (cDNA) hybridization probes using RNA-directed DNA polymerase isolated from avian myeloblastosis virus. Both of the probes selectively hybridized to RNA isolated from lactating mammary tissue, but not to poly(adenylic acid)-containing rat liver RNA. An analysis of the kinetics of hybridization of the cDNA derived from the 15S casein mRNA (cDNA12S) with their individual mRNA templates indicated that greater than 90% hybridization occurred over a R0t range of one and one-half logs with R0t 1/2 values of 0.0023 and 0.0032 mol s l.-1, respectively. Compared with the total RNA isolated from lactating mammary tissue, these values represented a 166- and 245-fold purification, respectively, of these individual mRNA fractions. Using the 15S casein mRNA as a template, two probes of different lengths and specific activities were synthesized. The deoxyribonucleotide and mRNA concentrations and the temperature of incubation were optimized to obtain either a high specific activity cDNA probe, 330 nucleotides long, which represented approximately 25% of the mRNA or a lower specific activity preparation containing some complete cDNA copies, 1300 nucleotides in length. The Tm of the longer cDNA15S-15S mRNA hybrid was 88.5 degrees C, while that of the short cDNA15S-RNA hybrid was 82.5 degrees C. Following this initial characterization, the cDNA15S probe was utilized for three separate determinations: (1) Analysis of the sequence divergence between mouse and rat casein mRNAs. It was observed that the rate of hybridization of heterologous rat cDNA15S-mouse casein mRNA was only 20% that of the homologous rat cDNA15S-rat casein mRNA hybridization. The resulting heterologous hybrid displayed approximately 17% mismatching compared with the homologous hybrid. (2) Determination of the gene dosage for casein mRNA in normal and malignant mammary cells. In this study, an analysis of the kinetics of hybridization of the high specific activity cDNA15S probe with an excess of DNA isolated from lactating mammary tissue, carcinogen-induced mammary tumors, or rat liver indicated that casein mRNA was transcribed from the nonlification or deletion was observed during tumor formation or the process of mammary differentiation. (3) Quantitation of casein mRNA sequences during normal mammary gland development. RNA excess hybridizations were performed using RNA extracted from either pregnant, lactating, or regressed rat mammary tissue. The concentration of casein mRNA molecules/alveolar cell was found to increase 12-fold from 5 days of pregnancy until 8 days of lactation and then declined to approximately 2% of the maximal level of 79 000 molecules/cell by 7 days after weaning. A coordinate increase was observed in casein mRNA sequences detected by cDNA hybridization and mRNA activity measured in a cell-free translation assay.     

A generally applicable improved method for preparation of single stranded cDNA probes from clones constructed in M13 vectors

A generally applicable simplified procedure for the preparation of radiolabeled cDNA hybridization probes from cDNA clones in M13 (M13mp8) bacteriophage vectors is described. A cDNA copy of the insert DNA is synthesized by controlled reaction with the Klenow fragment of E. coli DNA polymerase I, primed with oligo-dT or sequencing primer. The cDNA is separated from the recombinant phage DNA template by alkaline gel electrophoresis. Sensitivity of the cDNAs was tested by quantitative measurement of specific mRNAs in solution hybridization under RNA (R0t analysis) or cDNA (RNA titration) excess conditions. The procedure permits measurement of mRNA levels as small as 0.00001-0.00006% in total RNA preparation. Cellular accumulation of hormone-induced mRNAs for the milk proteins, whey acidic protein and epsilon-casein was also measured using the cDNAs.     

Differential expression of albumin and alpha-fetoprotein genes in fetal tissues of mouse and rat

We have carried out a comparative analysis of the expression of the albumin and alpha-fetoprotein (AFP) genes in yolk sac and liver at different stages of fetal and postnatal life, in rat and mouse. Albumin and AFP mRNA levels were examined in these tissues by R0t analysis of RNA excess-cDNA hybridization data and/or by Dot blot hybridization. In addition, size analysis of the mRNA sequences were performed by electrophoretic fractionation on agarose gels containing methylmercury hydroxide and hybridization to radioactive cloned rat and mouse albumin and AFP cDNA probes. In the mouse, substantial amounts of albumin mRNA molecules were found in the yolk sac at different stages of development, while minimal levels of albumin mRNA sequences were detected in the rat yolk sac. The mouse yolk sac albumin mRNA molecules were found to be associated with the polysomes and to be functional in cell-free translation systems. In the rat, a reciprocal relationship appears to exist between the concentrations of the two mRNAs in yolk sac and embryonic liver. In contrast, in the mouse a parallel increase in both albumin and AFP mRNA levels was found in these tissues during fetal development. These results suggest that the expression of the albumin and AFP genes may be subjected to different regulatory events in these two members of the Muridae family.     

Improved northern blot method for enhanced detection of small RNA

This protocol describes an improved northern blot method that enhances detection of small RNA molecules (<40 nt) including regulatory species such as microRNA (miRNA), short-interfering RNA (siRNA) and Piwi-interacting RNA. Northern blot analysis involves the separation of RNA molecules by denaturing gel electrophoresis followed by transfer and cross-linking of the separated molecules to nylon membrane. RNA of interest is then detected by hybridization with labeled complementary nucleic acid probes. We have replaced conventional UV-cross-linking of RNA to nylon membranes with a novel, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated, chemical cross-linking step that enhances detection of small RNA by up to 50-fold. This requires no specialized equipment, is relatively inexpensive and is technically straightforward. Northern blotting can be done in 2 d, but detection of a specific RNA can vary from minutes to days. Although chemical cross-linking takes longer (15 min to 2 h) than UV cross-linking, improved sensitivity means shorter periods of exposure are required to detect signal after hybridization.     

Molecular cloning of cDNAs for the nerve-cell specific phosphoprotein, synapsin I.

To provide access to synapsin I-specific DNA sequences, we have constructed cDNA clones complementary to synapsin I mRNA isolated from rat brain. Synapsin I mRNA was specifically enriched by immunoadsorption of polysomes prepared from the brains of 10-14 day old rats. Employing this enriched mRNA, a cDNA library was constructed in pBR322 and screened by differential colony hybridization with single-stranded cDNA probes made from synapsin I mRNA and total polysomal poly(A)+ RNA. This screening procedure proved to be highly selective. Five independent recombinant plasmids which exhibited distinctly stronger hybridization with the synapsin I probe were characterized further by restriction mapping. All of the cDNA inserts gave restriction enzyme digestion patterns which could be aligned. In addition, some of the cDNA inserts were shown to contain poly(dA) sequences. Final identification of synapsin I cDNA clones relied on the ability of the cDNA inserts to hybridize specifically to synapsin I mRNA. Several plasmids were tested by positive hybridization selection. They specifically selected synapsin I mRNA which was identified by in vitro translation and immunoprecipitation of the translation products. The established cDNA clones were used for a blot-hybridization analysis of synapsin I mRNA. A fragment (1600 bases) from the longest cDNA clone hybridized with two discrete RNA species 5800 and 4500 bases long, in polyadenylated RNA from rat brain and PC12 cells. No hybridization was detected to RNA from rat liver, skeletal muscle or cardiac muscle.     

Sequence complexity of cDNA transcribed from a diverse mRNA population

Mouse liver poly(A)+mRNA was reverse transcribed using oligo-p(dT) or random oligonucleotides as primers to yield cDNA about equal to the mass of the template RNA. The size profile of the oligo-p(dT)-primedd cDNA was similar to that of the template RNA. RNA or cDNA driven saturation annealing of labeled single copy genomic DNA (scDNA) showed that 2% of the scDNA was complementary in either case indicating the sequence complexity of cDNA was equivalent to that of the template mRNA. These results establish for the first time that cDNA represents essentially all of the sequence complexity of a diverse template RNA population in which individual mRNA species are present in vastly different concentrations. RNA driven hydridization of the cDNA showed that about 40% of the cDNA mass represents most of the sequence complexity of the template RNA. Also, kinetics of this hybridization indicate a complexity of 58,000 kb for the template RNA, a value similar to that obtained by scDNA hybridization. We conclude that appropriately characterized cDNA probes can be used to make valid qualitative and quantitative comparisons of the complex, infrequent class mRNAs of different cells and tissues.     

Quantitation of specific mRNA by RNA-RNA hybridization kinetics with single-stranded riboprobes

A quantitative procedure by a solution hybridization involving RNA-RNA hybridization kinetics was developed for measurement of specific mRNA accumulated in particular tissues and cells. For quantitating mouse beta-tubulin mRNA two types of riboprobes were prepared: one was a truncated RNA covering only the coding portion of beta-tubulin cDNA and the other was a non-truncated RNA covering the vector portion as well as the coding portion. These antisense RNAs were hybridized with mouse brain total cellular RNA, yielding heat-stable hybrids. Both the truncated and non-truncated antisense RNA probes showed similar hybridization kinetics. Hybridization of the sense RNA, consisting of the beta-tubulin coding portion, with the antisense RNA probe gave standards for determining the proportion of beta-tubulin mRNA in total brain RNA. By this method, the amounts of beta-tubulin mRNA included in the brains of 10- and 50-day-old mice were quantitated to be 0.0056 and 0.0011% of total RNA, respectively.     

Effect of unlabeled helper probes on detection of an RNA target by bead-based sandwich hybridization

Unlabeled helper oligonucleotides assisting a bead-based sandwich hybridization assay were tested for the optimal placement of the capture and detection probes. The target used was a full-length in vitro synthesized mRNA molecule. Helper probes complementary to regions adjacent to the binding site of the 5' end attached capture probe were found much more effective than helper probes targeting positions adjacent to the detection probe binding site. The difference is believed to be caused by a disruption of the RNA secondary structure in the area where the capture probe binds, thereby reducing structural interference from the bead. The use of additional helpers showed an additive effect. Using helpers at both sides of the capture and detection probes showed a 15- to 40-fold increase in hybridization efficiency depending on the target, thereby increasing the sensitivity of the hybridization assays. Using an electrical chip linked to the detection probe for the detection of p-aminophenol, which is produced by alkaline phosphatase, a detection limit of 2 x 10(-13) M mRNA molecules was reached without the use of a nucleic acid amplification step.     

Distribution of root mRNA species in other vegetative organs of pea (Pisum sativum L.)

Summary A cDNA library was prepared from, poly(A)⁺ RNA from roots of pea (Pisum sativum L.). Twenty five clones were selected by use of random numbers and used as probes on Northern blots to analyse the distribution of their corresponding mRNA species in other vegetative pea organs: leaf, stem and developing cotyledon. Fifteen cDNA inserts hybridised to single mRNA species, five hybridised to two mRNA species and one hybridised to five homologous mRNAs. Four cDNA clones (16% of those selected) gave no hybridization signals, indicating that the steady state levels of mRNAs were below the detection limit (i.e.less than 2.5 x 10^-5% of poly(A)⁺ RNA). Most of the root mRNAs were represented in all four pea organs as sequences of low and medium abundance. All but two cDNAs encoded mRNA species enhanced in root. However, cDNA clones appeared not to encode mRNA species expressed in a strictly organ-specific manner, as no mRNA unique to root was found. Thus, if organ-unique mRNA species are present, they are only present at a very low level of abundance in the poly(A)⁺RNA population.     

Localization of alpha-casein gene transcription in sections of epoxy resin-embedded mouse mammary tissues by in situ hybridization

The objective of our study was to evaluate the suitability of aldehyde-fixed, epoxy resin-embedded tissue for efficient and reproducible detection of casein mRNA in mouse mammary tissue by in situ hybridization. We used mouse alpha-casein-specific, 35S-labeled riboprobes generated from a Gemini-3 vector. Both complementary (anti-sense) and homologous (sense) RNA probes were utilized in our study (specific activity ranged from 5-7 x 10(8) cpm/micrograms). We tested the stability of newly synthesized [3H]-uridine-labeled RNA in tissue sections subjected to epoxy plastic solvents and found that no detectable loss of label occurred during preparation of semi-thin (1-2 micron) plastic sections for situ hybridization. In addition, it was possible to detect alpha-casein mRNA in deplasticized sections of mammary gland tissue taken from normal, pregnant, or lactating mice, pre-neoplastic mammary alveolar hyperplasias, explant cultures, and mammary tumors. A positive hybridization signal was consistently obtained in sections of mammary tissues where the estimated average copy number for total casein mRNA was greater than or equal to 250/cell. In mammary tumors, where the estimated casein mRNA content was much lower (less than 5/cell), our positive hybridization signal occurred in regions of the tumor that, in consecutive sections, stained positive for casein by immunoperoxidase. After formaldehyde-glutaraldehyde fixation, loss of hybridizable RNA from epoxy-embedded tissues and sections appears to be minimal. Image resolution was greatly enhanced over frozen or paraffin sections of mammary tissue. Non-specific binding of the radioactive probes was very low. Protease treatment of the sections was not necessary for detection of hybridizable signal.     

Elastin mRNA levels during foetal development of sheep nuchal ligament and lung. Hybridization to complementary and cloned DNA

Elastin mRNA levels were quantified in sheep nuchal ligament and lung during the latter half of foetal development with elastin-specific cDNA (complementary DNA) probes using both hybridization in solution (saturation analysis) and hybridization on a fixed support (Northern analysis). For the solution-hybridization studies, cDNA prepared from nuchal-ligament mRNA was enriched to 65% for elastin sequences by hybridizing it to its template at a R0t (mol X s X litre-1) value that included only the abundant class of mRNA sequences. Hybridization of this probe to RNA extracted from nuchal ligament between 70 and 138 days after conception demonstrated elastin sequences increased about 10-fold (from 0.047 to 0.438% of total RNA). In contrast, lung elastin mRNA levels increased only 3-fold (from 0.009 to 0.022% of total RNA) during the same period. Over this development period these values correspond to increases in the average number of elastin mRNA molecules from 950 to 20 000 molecules/ligament cell and from 130 to 330 molecules/lung cell. For Northern analysis, elastin mRNA was purified from near-term-sheep nuchal ligament on sucrose density gradients. Analysis of the translation products of this elastin mRNA showed that relative elastin precursor synthesis was at least 80% of total [3H]valine incorporation. The Mr of this elastin mRNA, determined by methylmercury-agarose-gel electrophoresis, was approx. 1.25 X 10(6). Northern hybridization of nuchal ligament and lung RNA to a [32P]cDNA probe, transcribed from this sucrose-gradient-purified elastin mRNA, confirmed the developmental changes in elastin mRNA levels detected by solution-hybridization techniques. The specificity of this method was confirmed by using a cloned elastin gene fragment. These studies demonstrate that elastin mRNA levels in organs such as nuchal ligament and lung increase with foetal development, but that there are significant differences in the average cellular elastin mRNA content of these two organs.     

Isolation and characterization of a cDNA coding for human myeloperoxidase

A cDNA encoding the carboxyl-terminal fragment of the human myeloperoxidase heavy chain was isolated and characterized. It was then used to determine the locations of the myeloperoxidase light and heavy chains in the polypeptide precursor. A cDNA library from poly(A)+ RNA from human leukemia HL-60 cells was constructed in pBR322 and screened by differential hybridization with enriched and depleted cDNA probes and then by hybridization with an oligonucleotide probe. A cDNA clone containing 1278 bp with an open reading frame of 474 bp and a 3' noncoding region of 804 bp was isolated. The amino acid sequence deduced from the nucleotide sequence consisted of 158 residues including a sequence of 14 amino acids known to be present in the heavy chain of the molecule. The cDNA also included a stop codon of TAG followed by a noncoding sequence that included a potential recognition site for polyadenylylation and a poly(A) tail. RNA transfer blot analysis with the cDNA probe indicated that myeloperoxidase mRNA was approximately 3.3 kb in length. In vitro translation of the mRNA selected by cDNA hybridization revealed preferential synthesis of a 74,000-Da polypeptide precursor that could be precipitated with anti-myeloperoxidase IgG. Antibodies specific for the heavy and light chains of myeloperoxidase were isolated from antiserum by affinity chromatography employing Sepharose columns covalently bound to the heavy or light chains. Antibodies specific for the light chain or the heavy chain readily precipitated the 74,000-Da precursor polypeptide. These results indicated that myeloperoxidase is synthesized as a single chain which undergoes processing into a light and heavy chain. Furthermore, the heavy chain of myeloperoxidase originates from the carboxyl terminus of the precursor polypeptide.     

Complex population of nonpolyadenylated messenger RNA in mouse brain

The complexity of nonadenylated mRNA [poly(A)-mRNA] has been determined by hybridization with single-copy DNA (scDNA) and cDNA. Our results show that poly(A)- and poly(A)+ mRNA are essentially nonoverlapping (nonhomologous) sequence populations of similar complexity. The sum of the complexities of poly(A)+ mRNA and poly(A)- mRNA is equal to that of total polysomal RNA or total mRNA, or the equivalent of approximately 1.7 x 10(5) different sequences 1.5 kb in length. Poly(A)- mRNA, isolated from polysomal RNA by benzoylated cellulose chromatography, hybridized with 3.6% of the scDNA, corresponding to a complexity of 7.8 x 10(4) different 1.5 kb sequences. The equivalent of only one adenosine tract of approximately 20 nucleotides per 100 poly(A)- mRNA molecules 1.5 kb in size was observed by hybridization with poly(U). cDNA was transcribed from poly(A)- mRNA using random oligonucleotides as primers. Only 1-2% of the single-copy fraction of this cDNA was hybridized using poly(A)+ mRNA as a driver. These results show that poly(A)- mRNA shares few sequences with poly(A)+ mRNA and thus constitutes a separate, complex class of messenger RNA. These measurements preclude the presence of a complex class of bimorphic mRNAs [that is, species present in both poly(A)+ and poly(A)- forms] in brain polysomes.