Abstract: A method for designing compounds specifically targeting RNA sequences, based on the discovery of short, specific sequences within RNA that are critical to function, using modeling of the compound to effect binding to the nucleotide sequences in the RNA in combination with secondary and/or tertiary structure associated with the minor groove of the RNA in the region of the critical sequences. In the preferred method, computer modeling is used along with analysis of the targeted RNA sequence to design molecules binding to the targeted RNA by covalent or hydrogen binding. Appropriate molecules are synthesized using known methodology that have the required structure and chemical characteristics to specifically bind the critical region of the RNA and thereby inhibit the function of the RNA. Molecules known to bind to RNA can also be modified using this method to increase specificity, and thereby decrease toxicity.

Abstract: A method for designing compounds specifically targeting RNA sequences, based on the discovery of short, specific sequences within RNA that are critical to function, using modeling of the compound to effect binding to the nucleotide sequences in the RNA in combination with secondary and/or tertiary structure associated with the minor groove of the RNA in the region of the critical sequences. In the preferred method, computer modeling is used along with analysis of the targeted RNA sequence to design molecules binding to the targeted RNA by covalent or hydrogen binding. Appropriate molecules are synthesized using known methodology that have the required structure and chemical characteristics to specifically bind the critical region of the RNA and thereby inhibit the function of the RNA. Molecules known to bind to RNA can also be modified using this method to increase specificity, and thereby decrease toxicity.

Abstract: Recombinant nucleic acids which encode aminoacyl-tRNA sythetases of pneumocystis origin or portions of such enzymes, have been isolated. These nucleic acids can be used to make expression constructs and transformed host cells for the production of pneumocystis aminoacyl-tRNA synthetases. They can also be used in the further isolation of nucleic acids related by DNA sequence similarities, which also encode pneumocystis aminoacyl-tRNA synthetases, or portions thereof. A further embodiment of the invention is antisense nucleic acid which can hybridize to the nucleic acid which encodes the aminoacyl-tRNA synthetase of pneumocystis. The invention also relates to enzymes, isolated and/or recombinant pneumocystis aminoacyl-tRNA synthetases. Antibodies which bind to these enzymes can be made and can be used in the purification and study of the enzymes.

Abstract: Recombinant nucleic acids which encode aminoacyl-tRNA sythetases of helicobacter origin have been isolated. These nucleic acids can be used to make expression constructs and transformed host cells for the production of helicobacter aminoacyl-tRNA synthetases. They can also be used in the further isolation of nucleic acids related by DNA sequence similarities, which also encode helicobacter aminoacyl-tRNA synthetases, or portions thereof. A further embodiment of the invention is antisense nucleic acid which can hybridize to the nucleic acid which encodes the aminoacyl-tRNA synthetase of helicobacter. The invention also relates to isolated and/or recombinant helicobacter aminoacyl-tRNA synthetases. Antibodies which bind to these enzymes can be made and can be used in the purification and study of the enzymes.

Abstract: Isolated and/or recombinant nucleic acids encoding mycobacterial methionyl-tRNA synthetase have been characterized. Recombinant DNA constructs and vectors having a sequence which encodes mycobacterial methionyl-tRNA synthetase have been made, and can be used for the construction of tester strains as well as for the production of isolated and/or recombinant methionyl-tRNA synthetases. These enzymes or portions thereof are useful in the biochemical separation of methionine and quantification of methionine or ATP, and for producing antibodies useful in the purification and study of the enzyme, for example. Host cells and methods useful for producing recombinant mycobacterial methionyl-tRNA synthetases are described, as are tester strains, which are cells engineered to rely on the function of the tRNA synthetase encoded by an introduced cloned gene.

Abstract: Isolated, recombinant nucleic acids which encode an isoleucyl-tRNA synthetase (IleRS) of human origin have been used to make expression constructs and transformed host cells for the production of a recombinant human IleRS. A recombinant enzyme has been purified, and is active in the specific aminoacylation of tRNA by isoleucine. Isolated, recombinant enzyme, and antibodies made specifically thereto, can be useful in assays to diagnose and monitor the autoimmune disease known as "antisynthetase syndrome." The essential isoleucyl-tRNA synthetases of microbes pathogenic in humans can be the targets of inhibitory agents having antimicrobial activity. A human isoleucyl-tRNA synthetase, isolated and purified, can be used to assess the toxic effect in humans of such an inhibitory agent in various biochemical activity assays. This human enzyme can also be expressed in "tester strains," whose cells rely upon the function of the human isoleucyl-tRNA synthetase for tRNA.sup.Ile charging.

Abstract: Isolated and/or recombinant nucleic acids encoding mycobacterial isoleucyl-tRNA synthetase have been characterized. Recombinant DNA constructs and vectors having a sequence which encodes mycobacterial isoleucyl-tRNA synthetase have been made, and can be used for the construction of tester strains as well as for the production of isolated and/or recombinant isoleucyl-tRNA synthetases. These enzymes or portions thereof are useful in the biochemical separation of isoleucine and quantification of isoleucine or ATP, and for producing antibodies useful in the purification and study of the enzyme, for example. Host cells and methods useful for producing recombinant mycobacterial isoleucyl-tRNA synthetases are described, as are tester strains, which are cells engineered to rely on the function of the tRNA synthetase encoded by an introduced cloned gene.

Abstract: Isolated and/or recombinant nucleic acids encoding mycobacterial seryl-tRNA synthetase have been characterized. Recombinant DNA constructs and vectors having a sequence which encodes mycobacterial seryl-tRNA synthetase have been made, and can be used for the construction of tester strains as well as for the production of isolated and/or recombinant seryl-tRNA synthetases. These enzymes or portions thereof are useful in the biochemical separation of serine and quantification of serine or ATP, and for producing antibodies useful in the purification and study of the enzyme, for example. Host cells and methods useful for producing recombinant mycobacterial seryl-tRNA synthetases are described, as are tester strains, which are cells engineered to rely on the function of the tRNA synthetase encoded by an introduced cloned gene.

Abstract: Isolated, recombinant nucleic acids which encode alanyl-tRNA synthetase (AlaRS) of human origin have been used to make expression constructs and transformed host cells for the production of recombinant human AlaRS. The recombinant enzyme has been purified, and is active in the specific aminoacylation of tRNA by alanine. The isolated, recombinant human AlaRS is also recognized by antibodies made by patients with the particular autoimmune disease known as "antisynthetase syndrome" in which the patients produce antibodies against the human alanyl-tRNA synthetase in their own cells. Thus, the isolated, recombinant enzyme, and antibodies made specifically thereto, can be useful in assays to diagnose and monitor this disease. The essential alanyl-tRNA synthetases of microbes pathogenic in humans can be the targets of inhibitory agents having antimicrobial activity.

Abstract: An analysis of LEU3, a leucine-specific regulatory locus encoding a factor for control of RNA levels of a group of leucine-specific genes, is provided.DNA sequence analysis of a clone of LEU3 shows that it contains an open reading frame of 886 amino acids. There are three regions of particular interest: a cluster of acidic amino acids that are located in the C-terminal half of the coding region, a region with a repeated cysteine motif, and a region of partial homology with MATalpha2. A LEU3-dependent DNA binding activity is demonstrated to interact with homologous portions of the 5'-region of LEU1 and LEU2.

Abstract: Disclosed is a method and linear DNA fragments for use in the deletion of a gene from a bacteria with a single step procedure that is applicable to any essential or nonessential gene which has been cloned. Chromosomal deletions are constructed by transformation of a cell strain with linear DNA fragments containing a locus for resistance to an antibiotic, or any other gene allowing for rapid phenotypic selection, flanked by sequences homologous to closely spaced regions on the cell chromosome on either side of the gene to be deleted, in combination with the immediate subsequent deletion or inactivation of the recA gene. By selecting for a double-crossover event between the homologous sequences, shown by the antibiotic resistance or other detectable phenotype, a chromosome disruption can be selected for which has effectively deleted an entire gene.

Abstract: Disclosed is a method for identifying and isolating gene sequences coding for polypeptide sequences which result in specific functional activities or phenotypes, which may then be synthesized and assembled to form new proteins. The assembled protein may contain only amino acid sequences essential for specific functions, have a new quaternary structure resulting from addition of an amino acid sequence with oligomeric activity, or exhibit multiple activities. Also disclosed is a method for identifying, analyzing and synthesizing amino acid sequences with oligomeric activity which may be used to enhance or add a new activity to a protein.In one embodiment, the piece with oligomeric activity is fused onto a polypeptide with one or more additional activities. In another embodiment, this polypeptide sequence is hybridized with a second polypeptide consisting only of the sequence with oligomeric activity.

Abstract: Chimeric peptides adapted for delivering neuropharmaceutical agents, such as neuropeptides into the brain by receptor-mediated transcytosis through the blood-brain barrier. The chimeric peptides include a peptide which by itself is capable of crossing the blood-brain barrier by transcytosis at a relatively high rate. The transportable peptide is conjugated to a hydrophilic neuropeptide which by itself is transportable only at a very low rate into the brain across the blood-brain barrier. The resulting chimeric peptide is transported into the brain at a much higher rate than the neuropeptide alone to thereby provide an effective means for introducing hydrophilic neuropeptides into the brain through the blood-brain barrier.

Abstract: An analysis of LEU3, a leucine-specific regulatory locus encoding a factor for control of RNA levels of a group of leucine-specific genes, is provided.DNA sequence analysis of a clone of LEU3 shows that it contains an open reading frame of 886 amino acids. There are three regions of particular interest: a cluster of acidic amino acids that are located in the C-terminal half of the coding region, a region with a repeated cysteine motif, and a region of partial homology with MATalpha2. A LEU3-dependent DNA binding activity is demonstrated to interact with homologous portions of the 5'-region of LEU1 and LEU2.

Abstract: A method for constructing polyproteins which can perform multiple sequential activities. A DNA sequence is constructed using genetic engineering techniques to insert sequences encoding the desired proteins into a plasmid, in the correct order, following a single promoter element and before a single stop codon. The reading frames of the mRNA sequences are phased so that a polyprotein with the desired activities, in the required order, is produced.Modified polyproteins can be produced by inserting or substituting amino acids into the mRNA sequence to create spaces between the individual proteins, to increase the stability of the total polyprotein, to change the spatial orientation of the individual proteins relative to each other and their substrates, and to modify the activity of the individual proteins.

Abstract: Disclosed is a method for the deletion of a gene from a bacteria using a single step procedure that is applicable to any essential or nonessential gene which has been cloned. The process requires the construction of chromosomal deletions by transformation of a cell strain with linear DNA fragments containing a locus for resistance to an antibiotic, or any other gene allowing for rapid phenotypic selection, flanked by sequences homologous to a closely spaced region on the cell chromosome. By selecting for a double-crossover event between the homologous sequences, shown by the antibiotic resistant or other detectable phenotype, a chromosome disruption can be selected for which has effectively deleted an entire gene.If the gene is essential to viability, the bacteria may be transformed with a plasmid which has a temperature-sensitive replicon and a wild-type allele of the essential gene. When E.

Abstract: 2'-deoxyadenosine 5'-0-(1-thiotriphosphate) (dATP(.alpha.-S)) was introduced into the 3'-ends of DNA restriction fragments with E. coli DNA polymerase I to give phosphorothioate internucleotide linkages. Such "capped " 3'-ends were found to be resistant to exonuclease III digestion. Moreover, the resistance to digestion is great enough that, under the conditions employed, just one strand of a double helix was digested by exonuclease III when "cap" was placed at only one end; when digestion was carried to completion, the production of intact single strands resulted. When digestion with exonuclease III was limited, and followed by S1 nuclease treatment, double stranded DNA fragments asymmetrically shortened from just one side were produced. In this way thousands of nucleotides can be selectively removed from one end of a restriction fragment.