Molecular cloning, expression, and pharmacological characterization of humEAA1, a human kainate receptor subunit.

Kainate is a potent neuroexcitatory agent; its neurotoxicity is thought to be mediated by an ionotropic receptor with a nanomolar affinity for kainate. In this report, we describe the cloning of a cDNA encoding a human glutamate ionotropic receptor subunit protein from a human hippocampal library. This cDNA, termed humEAA1, is most closely related to rat and human cDNAs for kainate receptor proteins and, when expressed in COS or Chinese hamster ovary cells, is associated with high-affinity kainate receptor binding. We have successfully established cell lines stably expressing humEAA1. This is the first report of establishment of stable cell lines expressing a glutamate receptor subunit. The relative potency of compounds for displacing [3H]kainate binding of humEAA1 receptors expressed in these stable cell lines was kainate > quisqualate > domoate > L-glutamate >> (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid > dihydrokainate > 6,7-dinitroquinoxaline-2,3-dione > 6-cyano-7-nitroquinoxaline-2,3-dione. Homooligomeric expression of humEAA1 does not appear to elicit ligand-gated ion channel activity. Nevertheless, the molecular structure and pharmacological characterization of high-affinity kainate binding of the humEAA1 expressed in the stable cell line (ppEAA1-16) suggest that the humEAA1 is a subunit protein of a human kainate receptor complex.

Molecular structure and pharmacological characterization of humEAA2, a novel human kainate receptor subunit.

A cDNA encoding a novel human glutamate receptor subunit protein was isolated from a human hippocampal library. This cDNA, termed humEAA2, is most closely related to rat cDNAs for kainate receptor proteins and, when expressed in COS cells, is associated with high affinity kainate receptor binding. The relative potency of compounds in displacing [3H]kainate binding was kainate greater than quisqualate greater than domoate greater than L-glutamate much greater than 6,7-dinitroquinoxaline-2,3-dione greater than dihydrokainate greater than 6-cyano-7-nitroquinoxaline-2,3-dione greater than (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid. Homomeric expression of humEAA2 does not appear to elicit ligand-gated channel activity. Nevertheless, the molecular structure and pharmacology of high affinity kainate binding suggest that humEAA2 is a novel subunit protein of a human kainate receptor complex.

Total chemical synthesis and expression in Escherichia coli of a maize glutathione-transferase (GST) gene.

We have constructed a totally synthetic gene encoding a maize glutathione S-transferase (GST I). This gene, composed of 1320 nucleotides (nt) (660 bp), was assembled from only 16 synthetic oligodeoxynucleotides (average length 83 nt), using an efficient one-step annealing/ligation protocol. Sequencing was performed to verify the authenticity of the final assembled gene. Significantly, not a single mutation was found in either of the two constructs sequenced, indicating a remarkably low mutation frequency. The synthetic gene was introduced into Escherichia coli where it was successfully expressed. The biological activity of the GST I enzyme produced in E. coli was monitored by assaying bacterial extracts for the ability to conjugate [14C]atrazine in the presence of glutathione. This biologically active synthetic GST1 gene can now be introduced into plants to assess its ability to confer tolerance to the triazine class of herbicides.

A centrifugation method for separation of plant viral genomic and subgenomic RNAs.

Using alfalfa mosaic virus (AMV) as a model, a simple method for separating plant viral genomic RNAs from their subgenomic counterparts was established. The method relies on sucrose gradient fractionation under carefully selected conditions of centrifugation and fraction collection. The RNA components are recovered in nearly quantitative yield and have full biological activity as measured by infectivity of the reconstituted RNAs in suitable protoplasts and plant hosts. The individual RNAs, on the other hand, show no such infectivity, indicating that the separation is indeed complete.

Rapid construction of large synthetic genes: total chemical synthesis of two different versions of the bovine prochymosin gene.

We have tested several different synthesis designs and assembly methodologies to develop an improved gene synthesis strategy which enables significantly longer nucleotide sequences to be easily constructed. This strategy, based in part upon our ability to synthesize high-quality extended-length oligodeoxynucleotides (over 100-mer in length), together with the use of chemical 5'-phosphorylation, and simplified low-melting-temperature agarose gel purification methods, combines ease, speed and high overall efficiency. We show that it is now feasible to synthesize routinely even long genes (at least 1-2 kb). To demonstrate this capability we have chemically synthesized and assembled two different versions of the gene encoding the bovine enzyme prochymosin (prorennin). One gene is essentially the natural bovine prochymosin gene sequence. In the second gene the codons have been optimized with regard to the codon bias of highly expressed yeast genes. Each synthetic gene was in excess of 1100 bp, yet they were assembled from only 13 or 14 pairs of complementary oligodeoxynucleotides (oligos), the average lengths of which were 87 and 82 bp, respectively. The 'mutation' rate was low enough to assess that more than 75% of all such oligo pairs (160-170 total nt) were error-free.

Nucleotide sequence of a protamine component CII gene of Salmo gairdnerii.

We have isolated, using nick-translated cloned protamine cDNA's as probes, several genomic clones containing protamine gene sequences from a Charon 4A library of Eco R1 digested rainbow trout (Salmo gairdnerii) DNA. One clone was chosen for detailed study and the 2.5 kbp Bam HI-Eco R1 restriction fragment containing the gene was subcloned in the plasmid pBR322. A 920 bp Bg1 II - Bam HI restriction fragment contains a sequence coding for protamine component CII as well as regions 5' and 3' to the mRNA coding portion. Present in the region 5' to the mRNA coding sequence are the promoter associated signals "TATA" box and "CAAT" box. The 5' untranslated region of the mRNA whose length and sequence were not established from the cDNA clones (1) was determined by nuclease mapping and starts within a sequence similar to the "capping signal" found in other genes. The protamine gene for CII contains no introns, a situation common to most histone genes, but, unlike the histone genes does not occur close to other protamine genes in a "cluster".

Molecular analysis of the protamine multi-gene family in rainbow trout testis.

We have synthesized a family of double-stranded cDNAs (ds cDNAs) using as a template the family of highly purified protamine mRNAs from rainbow trout testis. Individual pure protamine cDNA components were isolated by cloning this family of protamine ds cDNAs in a plasmid vector (pMB9). Clones containing protamine sequences were characterized by restriction mapping and by a positive hybrid-selected translation assay, which allowed us to correlate particular cDNAs with particular protein components. To allow more detailed comparisons, complete nucleotide sequences were determined for selected protamine clones. We have detected at least 5 distinctly different coding sequences, which nevertheless show at least 82% homology, and which have probably arisen by repeated gene duplication. These very highly conserved coding sequences do however contain a distinctly variable region near the 5'-end of the mRNA (N-terminus of the protein), corresponding to the major sites of serine phosphorylation. Since the amino acid sequences predicted by our DNA sequences were slightly different from those previously published (1), we have independently determined the amino acid sequences of protamine components CI, CII, CIII from our own source of trout testis. These new peptide sequences are completely consistent with those predicted by our nucleotide sequences. The 3'-untranslated regions of the protamine mRNAs are, surprisingly almost as highly conserved as the coding regions. Both coding and 3'-noncoding portions appear to be under a similar degree of selective pressure and evolutionary constraint to remain constant.

The purification of Q-containing tRNAs by periodate modification. Purification and nucleoside composition of two related Drosophila tyrosine tRNAs.

A simple procedure for the purification of Drosophila tRNAs which contain the hypermodified nucleoside Q was developed. The cis-diol group of Q renders it susceptible to attack by sodium periodate, with the resultant formation of a hydrophobic pyrrol ring structure. The increase in hydrophobicity after periodate modification, of those tRNA species which contain the Q nucleoside, causes them to be selectively retarded on benzoylated DEAE-cellulose or RPC-5 columns, and allows their isolation in essentially pure form. Utilizing this property, the Q-containing tyrosine tRNA from Drosophila, tRNATyr1delta, was purified. The non-Q-containing tyrosine tRNA, tRNA, tRNATyr1gamma, was also purified, and the nucleoside compositions of the two were determined and compared. In addition to the Q nucleoside, these tRNAs appear to differ by the presence (tRNATyr1delta) or absence (tRNATyr1gamma) of 5-methylcytidine. In all other respects, these tRNAs appear to be identical, and are probably products of the same gene which differ in their levels of post-transcriptional modification.

The role of the guanine insertion enzyme in O-biosynthesis in Drosophila melanogaster.

Drosophila tRNA can be guanylated by a crude enzyme from rabbit reticulocytes. Guanylating activity is also present in crude extracts of adult Drosophila. A major product of this reaction as well as several minor ones were resolved by RPC-5 chromatography. The main substrate of both the Drosophila and rabbit reticulocyte enzymes was the non-Q-containing aspartic acid tRNA, tRNA2gammaAsp. The QU-lacking (gamma) forms of asparagine, histidine and tyrosine tRNAs were also substrates and gave rise to the minor products of the reaction. In contrast, the Q- or Q*-containing (delta) forms of these tRNAs appear not to be substrates. The evidence strongly suggests that the guanyating enzyme is involved in Q biosynthesis and would be better termed a guanine replacement or pre-Q insertion enzyme.

A doubtful relationship between tyrosine tRNA and suppression of the vermilion mutant in Drosophila.

The conditions under which Drosophila melanogaster are grown markedly influence the amount of the hypermodified nucleoside Q found in certain tRNAs. This effect on Q biosynthesis was found in both the wild-type and the suppressor of sable [su(s)2] mutant. Suppressed vermilion flies [su(s)2v; bw] with 78% of the tyrosine tRNA in the Q-lacking (gamma) form had brown eyes indistinguishible from su(s)2v; bw flies with only 6% of the tyrosine tRNA in the gamma form. The proposal that this tRNA is a specific inhibitor or tryptophan pyrrolase in vermilion flies, and that its absence in su(s)2 flies is the mechanism of suppression is not consistent with these results. In addition, the su(s)2 locus does not seem to be primarily responsible for controlling Q biosynthesis as previously suggested.