Distinct molecular phenotypes in murine cardiac muscle development, growth, and hypertrophy.

The onset of cardiac hypertrophy is associated with characteristic changes in myocardial gene expression that are thought to recapitulate a developmental gene program. We report here the first gene expression profile of the murine myocardium, using a rapid method of quantitative expression analysis based on real-time analytical RT-PCR. This assay was used to measure expression levels of 29 genes in (1) late stage development as represented by day 1 neonatal ventricles, (2) normal cardiac growth in 3 and 18 month old mice, and (3) cardiac hypertrophy following pressure overload by aortic constriction. For males and females normal growth is not associated with differential expression although there is elevated expression of skeletal and smooth muscle actin mRNA's in males compared to females. Using normal adult ventricles as a reference, there are many qualitative and quantitative differences between the day 1 neonatal myocardium and experimental cardiac hypertrophy. These data suggest that the response to POL involves a subset of re-expressed developmental genes together with altered expression of genes not necessarily associated with cardiac development.

Synthesis and properties of cross-linked DNA duplexes.

A method has been devised to synthesize DNA duplexes with covalently connected strands. The structure of cross-linked duplexes was confirmed by a reaction with the restriction endonuclease AluI. The thermal stability of the resulting compounds was investigated.

Protein sorting by high-performance liquid chromatography. I. Biomimetic interaction chromatography of recombinant human deoxyribonuclease I on polyionic stationary phases.

Chromatographic separations can be tailored to exploit specific interactions between a stationary phase ligand and a protein structural feature of interest. Variations in this feature then form the basis for sorting a mixture of closely related proteins into defined subpopulations. This report describes the sorting of variants of recombinant human deoxyribonuclease I (rhDNase) that differ in the occurrence of deamidation at a single residue. rhDNase, an enzyme that non-specifically hydrolyzes DNA, is glycosylated and exhibits considerable charge heterogeneity owing to the sialylation and phosphorylation of its N-linked oligosaccharides. This heterogeneity obscures the relatively subtle differences between deamidated and intact rhDNase, preventing separation on this basis in conventional ion-exchange HPLC. Published structural information on bovine DNase reveals that the analogous labile asparagine residue is involved in DNA binding, so stationary phases containing polyanionic ligands mimicking nucleic acids were employed to separate the deamidation variants of rhDNase. Electrostatically immobilized DNA, a "tentacle" cation exchanger (TCX) and immobilized heparin columns all resolved the deamidated and intact forms of rhDNase when operated at pH 4.5. The ligands of the TCX and heparin columns are sufficiently long, flexible and polyanionic to interact with rhDNase in a manner similar to DNA and to sort rhDNase variants on the basis of the charge difference of a single residue involved in that interaction. A non-hydrolyzable double-stranded oligonucleotide analogue of DNA was also synthesized and immobilized to an HPLC support. This column, operated at pH 6, where rhDNase is active, resolved the two isomeric products of deamidation of rhDNase, i.e., variants of the enzyme containing either aspartate or isoaspartate in lieu of asparagine at the deamidation site in rhDNase. This is the first reported separation of intact variants of a glycoprotein differing on the basis of these isomeric products of deamidation through the common cyclic imide mechanism.

Perspectives on clinical trials for hypertonic saline/dextran solutions for the treatment of traumatic shock

Animal studies with hypertonic solutins suggest that they can achieve resuscitation of hypovolemic shock with extremely small volumes. Such small volume ressuscitation might be ideal in the field treatment of injured patients. Our studies to date, with 60 patients enterd into a prospective, randomized, placebo-controlled, and double-blind clinical trial, suggest that the use of a 7.5% NaCl/Dextran 70 solution increases blood pressures during transport. The solutions have been safe, and we have encoutered no adverse side effects from their use. Survival rates to date favor use of the solutions, but we do not have convincing statistical significance yet in that regard

Codon replacement in the PGK1 gene of Saccharomyces cerevisiae: experimental approach to study the role of biased codon usage in gene expression.

The coding sequences of genes in the yeast Saccharomyces cerevisiae show a preference for 25 of the 61 possible coding triplets. The degree of this biased codon usage in each gene is positively correlated to its expression level. Highly expressed genes use these 25 major codons almost exclusively. As an experimental approach to studying biased codon usage and its possible role in modulating gene expression, systematic codon replacements were carried out in the highly expressed PGK1 gene. The expression of phosphoglycerate kinase (PGK) was studied both on a high-copy-number plasmid and as a single copy gene integrated into the chromosome. Replacing an increasing number (up to 39% of all codons) of major codons with synonymous minor ones at the 5' end of the coding sequence caused a dramatic decline of the expression level. The PGK protein levels dropped 10-fold. The steady-state mRNA levels also declined, but to a lesser extent (threefold). Our data indicate that this reduction in mRNA levels was due to destabilization caused by impaired translation elongation at the minor codons. By preventing translation of the PGK mRNAs by the introduction of a stop codon 3' and adjacent to the start codon, the steady-state mRNA levels decreased dramatically. We conclude that efficient mRNA translation is required for maintaining mRNA stability in S. cerevisiae. These findings have important implications for the study of the expression of heterologous genes in yeast cells.

Production of native, correctly folded bovine pancreatic trypsin inhibitor by Escherichia coli.

A gene for bovine pancreatic trypsin inhibitor (BPTI) was fused to the coding sequence for the Escherichia coli alkaline phosphatase signal peptide and expressed in E. coli under the control of the alkaline phosphatase promoter. When induced in phosphate-depleted medium such cells produced a trypsin inhibitor that was indistinguishable from native, properly folded BPTI. In particular, the BPTI produced by E. coli had three disulfide bonds that appeared to be identical to those found in native BPTI, as assayed by sensitivity to iodoacetate, dithiothreitol, and urea. This expression/secretion system will make possible the production of variant BPTI molecules, thus allowing the perturbing effects of amino acid substitutions on BPTI folding, structure, and function to be assessed.

Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites.

A method is described for the efficient insertion of mutagenic oligodeoxynucleotide cassettes which allow saturation of a target amino acid codon with multiple mutations. Restriction sites are introduced by oligonucleotide-directed mutagenesis procedures to flank closely the target codon in the plasmid containing the gene. The restriction sites to be introduced are chosen based on their uniqueness to the plasmid, proximity to the target codon and conservation of the final amino acid coding sequence. The flanking restriction sites in the plasmid are digested with the cognate restriction enzymes, and short synthetic duplex DNA cassettes (10-25 bp) are inserted. The mutagenic cassette is designed to restore fully the wild-type coding sequence, except over the target codon, and to eliminate one or both restriction sites. Elimination of a restriction site facilitates selection of clones containing the mutagenic oligodeoxynucleotide cassette. To make the cassettes, single-stranded oligodeoxynucleotides and their complements are synthesized in separate pools containing different codons over the target. This method has been successfully applied to generate 19 amino acid substitutions at position 222 in the subtilisin protein sequence.

The tac promoter: a functional hybrid derived from the trp and lac promoters.

Two hybrid promoters that are functional in Escherichia coli have been constructed. These hybrid promoters, tacI and tacII, were derived from sequences of the trp and the lac UV5 promoters. In the first hybrid promoter (tacI), the DNA upstream of position -20 with respect to the transcriptional start site was derived from the trp promoter. The DNA downstream of position -20 was derived from the lac UV5 promoter. In the second hybrid promoter (tacII), the DNA upstream of position -11 at the Hpa I site within the Pribnow box was derived from the trp promoter. The DNA downstream of position -11 is a 46-base-pair synthetic DNA fragment that specifies part of the hybrid Pribnow box and the entire lac operator. It also specifies a Shine-Dalgarno sequence flanked by two unique restriction sites (portable Shine-Dalgarno sequence). The tacI and the tacII promoters respectively direct transcription approximately 11 and 7 times more efficiently than the derepressed parental lac UV5 promoter and approximately 3 and 2 times more efficiently than the trp promoter in the absence of the trp repressor. Both hybrid promoters can be repressed by the lac repressor and both can be derepressed with isopropyl beta-D-thiogalactoside. Consequently, these hybrid promoters are useful for the controlled expression of foreign genes at high levels in E. coli. In contrast to the trp and the lac UV5 promoters, the tacI promoter has not only a consensus -35 sequence but also a consensus Pribnow box sequence. This may explain the higher efficiency of this hybrid promoter with respect to either one of the parental promoters.

In vitro deletional mutagenesis for bacterial production of the 20,000-dalton form of human pituitary growth hormone.

The 20,000-dalton (20K) variant form of human growth hormone (hGH) present in extracts from pituitary glands differs from the major form of hGH (22K, 191 amino acids) by the deletion of amino acid residues 32-46. Using oligonucleotide-mediated mutagenesis, the DNA coding for these amino acids was deleted from the gene previously constructed by us (Goeddel et al., 1979) for microbial hGH production. The DNA to be deleted was looped out by the annealing of a synthetic oligodeoxyribonucleotide to the coding strand of the hGH gene contained on recombinant phage M13 mp8 DNA. Resulting heteroduplex structures were stabilized using primer-directed in vitro DNA synthesis in the presence of T4 DNA ligase. On transformation of Escherichia coli, these heteroduplex DNAs yielded phage whose genomes contained either the original or the partially deleted hGH gene, and genotypes were distinguished by in situ plaque hybridization with synthetic oligonucleotide probes. A gene with the correct deletion was used to express the short hGH variant in E. coli.

Portable Shine-Dalgarno regions: a system for a systematic study of defined alterations of nucleotide sequences within E. coli ribosome binding sites.

We have developed a gene expression system in Escherichia coli that contains a portable Shine-Dalgarno region. Transcription of this system is under the direction of a hybrid promoter (tacII) derived from trp and lac-UV5 promoter sequences which is followed by a region that encodes a portable Shine-Dalgarno region (PSDR). Using a series of synthetic PSDRs, we varied the four bases that follow the Shine-Dalgarno (SD) region. We found that the presence of four A residues or four T residues in this position gives the highest translational efficiency. The presence of four C residues reduces the translation efficiency by 50% as compared with PSDRs with A or T residues. The presence of four G residues following the SD region lowers the translational efficiency by at least 75% with respect to PSDRs with A or T residues.

A hybrid promoter and portable Shine-Dalgarno regions of Escherichia coli.

A gene expression system of Escherichia coli is described here that contains a portable Shine-Dalgarno region. Transcription of this system is under the direction of a hybrid promoter derived from trp and lac-UV5 promoter sequences which is followed by a region that encodes the portable Shine-Dalgarno (PSD) region. Using a series of synthetic PSD regions we varied the length (from 4 to 13 bases) of the Shine-Dalgarno region by increasing the number of bases on the mRNA that are complementary to the 3'-end of 16S rRNA. We found that increase of the Shine-Dalgarno region to 8 or 13 bases decreases the translation efficiency of the chimaeric leucocyte interferon messenger by 40%. We also varied in another series of PSD regions the four bases that follow the Shine-Dalgarno region. We found that the presence of four A residues or four T residues in this region results in the highest translation efficiency. The presence of four C residues reduces the translation efficiency by 50% as compared with PSD regions with A or T residues. The presence of four G residues following the Shine-Dalgarno region lowers the translation efficiency by 75% with respect to PSD regions with A or T residues.

Portable Shine-Dalgarno regions; nucleotides between the Shine-Dalgarno sequence and the start codon affect the translation efficiency.

This chapter describes a gene expression system of E. coli that contains a portable Shine-Dalgarno region. Transcription of this system is directed by a hybrid promoter derived from trp and lac-UV5 promoter sequences, which is followed by a region that encodes the portable Shine-Dalgarno region. We used a series of synthetic portable Shine-Dalgarno regions to vary the length (from 4 to 13 bases) of the Shine-Dalgarno by increasing the number of bases on the mRNA that are complementary to the 3' end of 16s rRNA. Increase in the Shine-Dalgarno region to 8 or 13 bases decreased the translation efficiency of the chimeric leukocyte interferon messenger by 40%. In another series of portable Shine-Dalgarno regions, we varied the four bases that follow the Shine-Dalgarno region. The presence of four A residues or four T residues in this region resulted in the highest translation efficiency. The presence of four C residues reduced translation efficiency by 50%, compared with A or T residues. The presence of four G residues after the Shine-Dalgarno region lowered translation efficiency by 75%, compared with A or T residues.

Conformational stability of alternating d (CG) oligomers in high salt solution.

The conformation of d (CG)n oligomers with n = 2,3 has been studied in aqueous solution in the presence of high salt concentration. A minimum n value of three is necessary to obtain a left-handed Z-helix. When d (CG)3 is flanked by three non Z-helicogenic alternating AT sequences the left-handed helix is unstable and a B-type conformation is obtained also at high salt concentration.

Synthesis of oligonucleotides on cellulose. Part II: Design and synthetic strategy to the synthesis of 22 oligodeoxynucleotides coding for gastric inhibitory polypeptide (GIP).

A solid-phase approach using trinucleotide building blocks and functionalized cellulose as solid support was used to synthesize the 22 oligodeoxynucleotides that constitute a totally synthetic gene coding for a 43 amino acid peptide. Gastric inhibitory Polypeptide. The overall strategy used for the design and synthesis of the oligomers is described.