Nucleic Acid Molecules Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translationof a DNA sequence determined as above.
Using the information provided herein, such as the nucleotide sequence in SEQ ID NO: 1, a nucleic acid molecule of the present invention encoding a antimicrobial peptide polypeptide may be obtained using standard cloning and screening procedures,such as those for cloning cDNAs using mRNA as starting material.
By a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at leastabout 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide.
Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3' terminal poly(A) tract of the antimicrobial peptide cDNA shown in SEQ ID NO: 1), or to a complementary stretch of T (or U) resides, would not beincluded in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g.,practically any double-stranded cDNA clone).
Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) shown in SEQ ID NO: 1; DNA molecules comprising the coding sequence for the mature antimicrobial peptide protein; and DNAmolecules which comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the antimicrobial peptide protein.
The determined nucleotide sequence of theantimicrobial peptide cDNA of SEQ ID NO: 1 contains an open reading frame encoding a protein of about 64 amino acid residues, a predicted leader sequence of about 23 amino acid residues, and a deduced molecular weight of about 7.3 kDa.
More potential for toxicity since fungi share the same mechanisms to synthesize proteins and nucleic acids as other Eukaryotes. Many target the of fungal membranes. When synthesis is interrupted, the membrane becomes excessively permeable, killing the cell.
Nucleoside and Nucleotide Analogs: are synthetic compounds which resemble nucleosides, but have an incomplete or abnormal -ribose or ribose group. Once activated by , the drug competes with normal nucleotides for incorporation into viral DNA or RNA. Incorporation into the growing nucleic acid chain results in irreversible association with the viral polymerase and chain termination.
As indicated, nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
Interference with nucleic acid synthesis (RNA and DNA), whichexploits differences between RNA polymerases and DNA replicationstrategies in bacteria and eucaryotes.
By "isolated" nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes ofthe present invention.
Antibiotics like sulfonamides and trimethorim interfere with folate metabolism by blocking enzymes required for the synthesis of tetrahydrofolate; an enzyme needed by bacterial cells for the synthesis of folic acid production of DNA and RNA and amino acids.
As indicated, nucleic acid molecules of the present invention which encode a antimicrobial peptide polypeptide may include, but are not limited to those encoding the amino acid sequence of the mature polypeptide, by itself; the coding sequencefor the mature polypeptide and additional sequences, such as those encoding the about 23 amino acid leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3, sequences, such as the transcribed, non-translated sequences that play a role intranscription, mRNA processing, including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additionalfunctionalities.
Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having antimicrobial peptide activity include, interalia, (1) isolating the antimicrobial peptide gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the antimicrobial peptide gene, asdescribed in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); and Northern Blot analysis for detecting antimicrobial peptide mRNA expression in specific tissues.