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.     

Synthesis of alpha subunit of human chorionic gonadotrophin by presumptive HeLa cells

Summary Several cell lines, originally thought to be derived from a human placenta at term but possibly HeLa-contaminated, have been studied. These cells secrete a protein indistinguishable immunochemically from the alpha subunit of chorionic gonadotropin but not the beta subunit of chorionic gonadotropin or placental lactogen. Complete chorionic gonadotropin was detected but amounted to less than 1% of the level of the alpha subunit. The cells also produce an alkaline phosphatase similar to placental alkaline phosphatase in immunochemical, gel-electrophoretic, and heat-denaturation properties. They induce tumor growth when inoculated into nude mice. These cells are aneuploid and have a model chromosome number of 66. The common HeLa karyologic markers, designated 1, 2, and 3, and A-type glucose-6-phosphate dehydrogenase are present in these cells. HeLa cells have not previously been shown to secrete theα subunit of hCG.     

Properties of biologically active messenger RNA from human placenta. Cell-free synthesis of two immunoreactive forms of placental lactogen.

In order to understand better the regulation of human placental proteins the activity of placental lactogen messenger RNA has been examined. Total RNA was extracted from normal term placentas and purified by chromatography on oligo(dT)-cellulose. The poly(A)-containing fraction stimulated amino acid incorporation 5- to 10-fold in wheat germ cell-free extracts, and immunoprecipitation of the translation products with antiserum directed against human placental lactogen (hPL) suggests that about 2% of the peptides contain hPL determinants. Analysis of the material precipitated with hPL antiserum by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed two major species, one co-migrating with hPL and the other migrating slightly slower than hPL. On DEAE-cellulose chromatography the former material eluted close to authentic hPL while the latter material eluted at higher ionic strength than hPL, indicating a difference in net charge of these two species. Tryptic peptide analysis of the large material and authentic hPL shows marked similarities in the primary structure of these two proteins. The slower migrating peptide has an apparent molecular weight about 3000 larger than hPL and thus may represent a precursor molecule. Both cell-free products could be competed out of immunoprecipitates by a large excess of authentic hPL, confirming their immunologic similarities. Centrifugation of the placental poly(A)-containing RNA through aqueous glycerol gradients indicates that the hPL mRNA sediments at about 14 S.     

Distinct lectin activities from six species of cellular slime molds.

Extracts of cohesive cells of four species of cellular slime mold, D. mucoroides, D. purpureum, D. rosarium and P. violaceum agglutinate erythrocytes in a manner that is similar to that previously observed with extracts of D. discoideum and P. pallidum. We determined inhibitory activity of a series of sugars on the agglutination activity of each of these extracts, using both semiquantitative and quantitative agglutination assays. The inhibitory potency of this series of sugars was distinct for each extract, although only slight differences were found between several species, especially D. discoideum and P. violaceum. A possible role of these agglutinins in species-specific cell cohesion is considered.     

Regulation of casein messenger RNA during the development of the rat mammary gland.

Casein mRNA was isolated and partially purified from RNA extracts of rat lactating mammary glands and translated in a teterologous cell-free protein synthesizing system derived from wheat germ. Casein mRNA activity was assayed by immunoprecipitation using a specific antiserum prepared against a mixture of the purified rat caseins. Properties of rat casein mRNA were examined using a variety of sizing techniques, including chromatography on Sepharose 4B, sedimentation on sucrose gradients after heat denaturation, and electrophoresis on 2.5% agarose gels in 6 M urea. Casein mRNA activity was found in an 8-16S region after gradient centrifugation with the peak occurring at 10.5 S. In addition, the binding of rat casein mRNA to dT-cellulose was examined. Only 40% of the total casein mRNA activity was selectively retained. A partial purification of casein mRNA was accomplished by a combination of these sizing and affinity chromatography techniques. In the purified preparations casein mRNA activity comprises approximately 90% of the total mRNA activity. Characterization of this material by agarose gel electrophoresis revealed two main bands of RNA at approximately 12 and 16 S, both containing casein mRNA activity. These mRNAs were of the correct size to code for two of the principal rat caseins of approximately 25,000 and 42,000 molecular weights. Casein mRNA and total mRNA activities were then compared in total RNA extracts at various stages of normal mammary gland development in the rat, i.e. during pregnancy, lactation, and involution following weaning. A selective induction of casein mRNA activity compared to total mRNA activity was found to occur during pregnancy and lactation. Moreover, a selective loss of activity was also observed during mammary gland involution. A surprisingly high level of casein mRNA activity was found in RNA extracts from early and midpregnant mammary glands.     

Retention of endocrine function by an insulin-secreting pancreatic islet cell tumour from Syrain hamster through serial transplantation in nude mice.

An insulin-secreting islet cell tumour of the Syrian hamster has been transplanted serially in the congenitally immune-deficient nude mouse, in order to test the potential usefulness of this mouse mutant as a graft carrier of heterologous tumours with stable differentiated phenotypes. The incidence of tumour growth was very high, and the hamster tumour retained its functional and histologic characteristics during consecutive passages in nude mice. These results show that nude mice may be useful carriers of differentiated tumours from non-inbred species including man, and for the isolation of cell lines from such tumours.     

A quantitative description of normal AV nodal conduction curve in man.

The AV nodal conduction curve generated by the atrial extrastimulus technique has been described only qualitatively in man, making clinical comparison of known normal curves with those of suspected AV nodal dysfunction difficult. Also, the effects of physiological and pharmacological interventions have not been quantifiable. In 50 patients with normal AV conduction as defined by normal AH (less than 130 ms), normal AV nodal effective and functional refractory periods (less than 380 and less than 500 ms), and absence of demonstrable dual AV nodal pathways, we found that conduction curves (at sinus rhythm or longest paced cycle length) can be described by an exponential equation of the form delta = Ae-Bx. In this equation, delta is the increase in AV nodal conduction time of an extrastimulus compared to that of a regular beat and x is extrastimulus interval. The natural logarithm of this equation is linear in the semilogarithmic plane, thus permitting the constants A and B to be easily determined by a least-squares regression analysis with a hand calculator.