Relations between family environment and adjustment outcomes in young adults with spina bifida.

Thirty-two young adults with spina bifida completed a questionnaire (Family Environment Scale) assessing their perceptions of family social environment while growing up. Additionally, subjects responded to a structured interview addressing their current employment status, residential situation, level of community mobility, and extent of social activity. Multiple logistic regression analyses were used to assess the relation between family environment and adjustment as a young adult. With this limited sample, results indicated that perceived family environment explained variance in employment, community mobility, and social activity as an adult, even beyond that explained by lesion level and intelligence. Regression coefficients showed positive relations between perceived family encouragement of independence and achievement and young adult outcomes. In contrast, perceived moral/religious emphasis of the family and degree of family involvement with intellectual/cultural activities evidenced negative relations with the measures of young adult adjustment.

A monoclonal antibody against the c-erbB-2 protein enhances the cytotoxicity of cis-diamminedichloroplatinum against human breast and ovarian tumor cell lines.

A monoclonal antibody (TAb 250) specific to an extracellular epitope of the c-erbB-2 protein (gp185) inhibited the in vitro proliferation of human breast tumor cell lines that overexpress c-erbB-2 in a dose-dependent manner. Treatment of cells with combinations of cis-diammedichloroplatinum (CDDP) and TAb 250 resulted in a significantly enhanced cytotoxic effect. This synergistic cytotoxicity was apparent over a wide range of antibody concentrations (200 pg/ml-100 micrograms/ml) including concentrations that showed no inhibitory effect alone. TAb 250 did not increase the cytotoxic effect of CDDP in a cell line exhibiting no detectable level of gp185. Athymic mice bearing s.c. xenografts of human tumor cells expressing high levels of gp185 showed a greatly enhanced inhibition of tumor growth when treated with TAb 250 and CDDP compared to treatment with the antibody or CDDP alone. This effect was specific inasmuch as TAb 250 did not enhance the growth-inhibitory effect of CDDP on tumor xenografts which were not expressing gp185.

Molecular cloning and sequence determination of rat preproenkephalin cDNA: sensitive probe for studying transcriptional changes in rat tissues.

A cDNA probe was prepared to investigate the regulation of proenkephalin biosynthesis in the rat. This was necessary because human and bovine proenkephalin cDNA were not sensitive enough for the accurate detection of preproenkephalin mRNA in tissues that contain low copy numbers of this message, such as the adrenal gland. The rat probe was prepared in the following manner. Preproenkephalin mRNA was enriched by sucrose gradient centrifugation of poly(A)-containing mRNA from rat brain and was used as a template for double-stranded cDNA synthesis. The resulting cDNA was inserted into the plasmid pBR322, and recombinant plasmids were used to transform Escherichia coli RR1 cells. A synthetic oligodeoxyribonucleotide (30 bases long) with a sequence that had previously been shown to be identical in bovine and human preproenkephalin cDNA was prepared to screen the clone bank. The plasmid with the longest cDNA insert (about 1200 bases) from the positive clones was isolated, and the sequence of the entire protein coding region was determined. Like the bovine and human gene products, rat preproenkephalin contains four [Met]enkephalin sequences and one copy each of [Leu]enkephalin, [Met]enkephalin-Arg6-Gly7-Leu8, and [Met]enkephalin-Arg6-Phe7. Rat preproenkephalin is 80% and 83% homologous to the bovine and human forms, respectively, at the nucleotide level and is 82% homologous to both species at the amino acid level. Rat preproenkephalin contains 269 amino acid residues, making it larger than the human (267 residues) and bovine (263 residues) precursors. The sensitivity for detection of rat preproenkephalin mRNA with the rat cDNA was several times greater than with the corresponding cDNAs from bovine and human sources.

In vivo strain patterns in the four major canine knee ligaments.

Using mercury gauges, we measured strains in vivo in the four major ligaments of the canine knee joint as the tibia was loaded in valgus or varus at fixed angles of knee flexion. Free axial rotation of the tibia on the femur was allowed. Forces up to 78.4 N were applied to the tibia, producing moments of approximately 9 N-m. We found that with valgus loading, significant strains were observed in the medial collateral ligament at extension. At 45 degrees of flexion, the medial collateral, posterior cruciate, and anterior cruciate were strained. At 90 degrees of flexion, all four ligaments were strained. With varus loading, significant strains were found in the lateral collateral and anterior cruciate at extension. The lateral collateral and anterior cruciate ligaments were strained at 45 degrees of flexion. At 90 degrees of flexion, the lateral collateral, anterior cruciate, and posterior cruciate ligaments were strained. With valgus loading, the tibia rotated internally and the degree of axial rotation increased with flexion. External rotation of the tibia resulted from varus loading, and was relatively constant through the range of flexion. Thus when axial rotation is allowed, stability of the knee in response to valgus and varus loads is maintained by the cruciates as well as the collaterals, and the role of the cruciates increases with flexion and axial rotation.

Cloning and sequence analysis of cDNA for the precursor of human growth hormone-releasing factor, somatocrinin.

Molecular cloning has established the primary structures of two precursors of the human pancreas growth hormone-releasing factor (hpGRF-44), somatocrinin. Both polypeptides contain the sequence of hpGRF-44 flanked by basic processing sites. Furthermore, the precursors include a putative signal sequence and a carboxyl-terminal amidation signal for hpGRF-44. The two forms of mRNA code for pre-pro-GRF-107 and pre-pro-GRF-108. Pre-pro-GRF-108 differs from pre-pro-GRF-107 by the insertion of a serine in the carboxyl-terminal portion of the precursor. In vitro translation of tumor poly(A)+ RNA followed by immunoprecipitation with GRF-specific antiserum and gel electrophoresis showed the molecular weight of preprosomatocrinin to be approximately 13,000, which is in good agreement with the molecular weight deduced from the sequences of the cDNA clones.

Cloning of total mRNA populations from adult and embryonic mice.

Total clone banks of cDNAs synthesized from poly(A)-RNA obtained from three stages of the developing mouse were constructed. The stages chosen were 13-day-old embryo, neonatal, and fully grown adult. To have as complete a bank as possible, large numbers of individual clones were generated approximately 400,000 for the 13th day embryo and neonatal mouse and approximately 610,000 for the adult bank. In each case the clone bank was constructed by inserting double stranded cDNA into the PstI site of pBR322 by the "G-C tailing" method. Sequences cloned in this way could be separated from the plasmid host DNA by treatment of the resultant total chimeric plasmid population with PstI. Aliquots of the cloned cDNA material were labeled with 32P by "nick translation" using Escherichia coli DNA polymerase I for the preparation of hybridization probes. Back-hybridization of these probes to the total clone banks allowed the determination of the sequence diversity among the above three very different developmental stages. The use of such clone banks should allow the identification of developmental stage specific mRNAs.

Transformation of E. coli using homopolymer-linked plasmid chimeras.

A number of parameters were explored to increase the transformation efficiency of E. coli with pBR322/eukaryotic DNA chimera, formed via d(A) . d(T) and d(G) . d(C) homopolymer tails. Of the E. coli strains analyzed, E. coli strain RR1 was the most efficient bacterial host. A clear optimum of nucleotide tail length existed for both types of homopolymer. The optimum hybridization temperature for chimera formation was found to be approx. 57 degrees C. In the case of d(A) . d(T)-linked chimeras, 30 min was sufficient for optimum chimera formation. In contrast, d(C) . d(G)-linked chimeras required up to 2 h to give the best yields (as measured by transformation efficiency). Other minor factors affecting the transformation process are also explored and discussed.

On the cloning of eukaryotic total poly(A)-RNA populations in Escherichia coli.

A total clone bank of cDNAs synthesized from mouse liver poly(A)-RNA was constructed in Escherichia coli K-12 RR1 using the plasmid pBR322. Sequences of cDNAs were inserted into the PST-I site of pBR322 by the "G-C-tailing" method. Bulk preparations of these cDNA sequences were obtained by treatment of the resultant total hybrid plasmid population with PST-I. Aliquots of this cDNA material were then labeled with 32P by "nick translation" using E. coli DNA polymerase I for the preparation of hybridization probes. Experiments involving the back-hybridization of these probes to the total hybrid-plasmid clone bank population revealed that virtually all of the liver poly(A)-RNA sequences were represented in the total clone bank. The long term stability of these sequences residing in RR1 host cells was then examined through extensive serial passage of the initial total clone cell population culture. The results showed: 1) that the small percentage of natural pBR322 molecules (containing no cDNA inserts) usually present in such total clone bank preparations do not outgrow the respectively hybrid specie of plasmids in these populations: 2) few, if any, cDNA sequences are completely lost; and 3) some redistribution of the abundant and more unique DNA sequences probably does occur. The use of total clone hybrid-plasmid populations (constructed from poly(A)-RNA isolated from different tissues) described here should allow the identification of tissue-specific RNA sequences through the use of cross-hybridizaton techniques.

Synthesis, cloning, and identification of DNA sequences complementary to mRNAs for alpha and beta subunits of thyrotropin.

Double-stranded cDNA sequences were synthesized, by using as templates mRNA for alpha and beta subunits of thyrotropin purified from mouse thyrotrophic pituitary tumours and cloned in Escherichia coli RR1 by insertion in the Pst I site of the bacterial plasmid pBR322 by use of poly(dA) x poly(dT) homopolymeric extensions. Plasmids containing inserted cDNA sequences were selected by resistance to tetracycline and sensitivity to ampicillin; those containing thyrotropin cDNA sequences were identified by colony hybridization with an 125I-labeled mixture of alpha and beta thyrotropin mRNAs. Plasmids carrying either alpha or beta thyrotropin cDNA sequences were further identified by hybridization to highly purified 125I-labeled alpha or beta thyrotropin mRNA, respectively. Two plasmids, one containing a 400-nucleotide alpha thyrotropin cDNA insert and the other containing a 710-nucleotide beta thyrotropin cDNA insert, were purified and characterized by restriction endonuclease digestions. Plasmid [32P]DNA containing either alpha or beta thyrotropin cDNA was then used as a hybridization probe for further characterization of alpha and beta thyrotropin mRNA from the mouse thyrotropic tumor. RNA was fractionated by agarose gel electrophoresis under denaturing and nondenaturing conditions and transferred to diazobenzyloxymethyl-paper. alpha thyrotropin mRNA of two sizes, 650 and approximately equal to 1500 nucleotides long, were identified. The larger alpha thyrotropin mRNA appeared to have marked secondary structure in its native form in contrast to the 650-nucleotide alpha thyrotropin mRNA. However, only one form of beta thyrotropin mRNA was detected with an apparent size of 710 nucleotides. We have successfully cloned and identified alpha and beta thyrotropin cDNA sequences in bacterial plasmids and used them to identify a second form of alpha thyrotropin mRNA.

Protein patterns of early mouse embryos during development.

Using high resolution two-dimensional polyacrylamide gel electrophoresis, the major protein species (labeled in vitro with 35S-methionine) of mouse embryos were examined starting with the unfertilized egg up to the blastocyst stage (fourth day of development). The analysis was then continued using in vitro culture techniques up to the 10th equivalent gestation day. At all periods of development distinct protein changes could be seen. However, major alterations in the protein synthesis pattern were noted between the 2nd and 3rd day in vivo and around the 8th equivalent gestation day in vitro. A complete series of gels is presented such that proteins can be easily identified in terms of their molecular weights and isoelectric points.

Increased amplification of pBR322 plasmid deoxyribonucleic acid in Escherichia coli K-12 strains RR1 and chi1776 grown in the presence of high concentrations of nucleoside.

When pBR322 plasmid-harboring Escherichia coli strains RR1 or chi1776 were grown in the presence of 1 mg of uridine or cytidine per ml and later treated with chloramphenicol, as much as three times more plasmid deoxyribonucleic acid was recovered than would normally be obtained by routine plasmid amplification procedures.

Factors affecting the transformation of Escherichia coli strain chi1776 by pBR322 plasmid DNA.

The susceptibility of E. coli strain chi1776 to transformation by pBR322 plasmid DNA was examined and optimized. Maximum transformation to tetracycline (Tc) resistance was achieved when cells were harvested from L broth at 5.0--6.0 . 10(7) cfu/ml, followed by washing twice in cold 0.1 M NaCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6. Cells grown in the presence of D-cycloserine (Cyc) rather than nalidixic acid (Nx) transformed markedly better. The presence of 5 mM Mg2+ ions in washing and CaCl2 solutions stimulated transformation about 2-fold. Optimal conditions for transformation included a pH range of 7.25-7.75 and a cell-to-DNA ratio of about 1.6 . 10(8) cfu/ng plasmid DNA. The frequency of transformation was highest when cells were exposed to 100 mM CaCl2 in 250 mM KCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6, before mixing with DNA. A 60 min incubation period for cell + DNA mixtures held on ice produced the maximum number of Tcr transformants. In our hands, heat shocks at 37 degrees C or 42 degrees C for various times all decreased transformation to about one-half of optimal levels. Furthermore, the recovery of transformants was best when cell + DNA mixtures were plated on precooled (4 degrees C) Tc agar plates. The efficiency of plating was optimum when only 5 microliter of cell + DNA mixture was spread per plate, suggesting that non-viable background chi1776 cells on selective medium inhibited the recovery of transformants. It was also found that the presence of linear DNA molecules in cell + DNA mixtures markedly inhibited the transformation of chi1776 by pBR322 plasmid DNA. On the basis of these findings, a new procedure for the plasmid-specific transformation of E. coli chi1776 by pBR322 plasmid DNA is proposed. The use of this technique has allowed us to attain transformation frequencies in excess of 10(7) transformants/microgram pBR322 plasmid DNA.

The ovalbumin gene. Insertion of ovalbumin gene sequences in chimeric bacterial plasmids.

Double-stranded ovalbumin DNA was amplified and purified by the cloning of bacterial transformants. The double-stranded DNA was synthesized from a complete complementary DNA transcript of ovalbumin mRNA using Escherichia coli DNA polymerase I and the self-priming ability of the initial transcript. After S. nuclease treatment, poly(dA) was added to the 3' termini with terminal deoxynucleotidyltransferase and the ovalbumin gene was hybridized to a linear plasmid DNA, pMB9, containing 3'-poly(dT) termini. This hybrid molecule was used to transform the E. coli strain X1849. The cloned transformants contained from 30 to 53% of the complete ovalbumin DNA as determined by hybridization with full length cDNA. The length of the inserts was confirmed by treatment of the isolated plasmids with the restriction enzyme Hha I. Separation of the fragments by agarose gel electrophoresis showed that the amount of inserted DNA in clones tested varied from 680 to 1090 base pairs.