Thomas M. Shinnick

Immunogenic structure of the influenza virus hemagglutinin

We chemically synthesized 20 peptides corresponding to 75% of the HA1 molecule of the influenza virus. Antibodies to the majority (18) of these peptides were capable of reacting with the hemagglutinin molecule. These 18 peptides are not confined to the known antigenic determinants of the hemagglutinin molecule, but rather are scattered throughout its three-dimensional structure. In contrast, antibody raised to intact hemagglutinin did not react with any of the 20 peptides. Taken together these results suggest that the immunogenicity of an intact protein molecule is not the sum of the immunogenicity of its pieces.

Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis

The emergence of multidrug-resistant (MDR) tuberculosis (TB) highlights the urgent need to understand the mechanisms of resistance to the drugs used to treat this disease. The aminoglycosides kanamycin and amikacin are important bactericidal drugs used to treat MDR TB, and resistance to one or both of these drugs is a defining characteristic of extensively drug-resistant TB. We identified mutations in the −10 and −35 promoter region of the eis gene, which encodes a previously uncharacterized aminoglycoside acetyltransferase. These mutations led to a 20–180-fold increase in the amount of eis leaderless mRNA transcript, with a corresponding increase in protein expression. Importantly, these promoter mutations conferred resistance to kanamycin [5 μg/mL < minimum inhibitory concentration (MIC) ≤40 μg/mL] but not to amikacin (MIC <4 μg/mL). Additionally, 80% of clinical isolates examined in this study that exhibited low-level kanamycin resistance harbored eis promoter mutations. These results have important clinical implications in that clinical isolates determined to be resistant to kanamycin may not be cross-resistant to amikacin, as is often assumed. Molecular detection of eis mutations should distinguish strains resistant to kanamycin and those resistant to kanamycin and amikacin. This may help avoid excluding a potentially effective drug from a treatment regimen for drug-resistant TB.

Identifying the protein products of brain-specific genes with antibodies to chemically synthesized peptides

From the nucleotide sequences of three cDNA clones of rat brain-specific mRNAs, we deduced the partial amino acid sequences of two previously unknown proteins. We raised antisera to synthetic peptides mimicking short regions of these putative brain-specific proteins, and used these sera in immunocytochemical studies to localize each protein in the brain. One protein is found in large neurons throughout the brain, asymmetrically distributed toward the dendritic pole of the cell cytoplasm, suggesting involvement in the synthesis and/or directional transport of dendritic substances. The sequence of the second protein contains pairs of basic residues similar to precursors for neurotransmitters. The protein is located in (and may be the precursor for the neurotransmitter of) a novel fiber network of major extent with ramifications in cerebellum, hippocampus, and cortex, and in cell bodies in the brain stem, hypothalamus, and caudate nucleus. Our approach provides a direct method for characterizing rare brain molecules which may not have been anticipated and for which there are no known functional assays.

A gene,imz, affecting the pH sensitivity of zygote formation inPhysarum polycephalum

Mating inPhysarum polycephalum involves the fusion of two haploid amoebae and the differentiation of the resulting diploid zygote into a multinucleate plasmodium. Mating proceeds optimally with amoebae growing on an agar medium at pH 5.0. At pH 6.2, the amoebae still grow normally, but mating is completely blocked. The barrier at pH 6.2 is not in the differentiation step, since preformed diploids readily convert to plasmodia at this pH. The barrier can be overcome by raising the ionic strength of the agar medium; the effect, moreover, is not ion-specific. We have discovered a genetic locus,imz (ionicmodulation of zygote formation), that affects the upper pH limit for mating; the respective limits associated with the two known alleles,imz-1 andimz-2, are pH 5.6 and pH 6.0 at low ionic strength. Animz-1×imz-2 mating displays the pH 6.0 limit;imz-2 is therefore “dominant”. We suggest that this new gene affects a cell component that is exposed to the exterior of the amoeba and is involved in the fusion step of mating.

Mutants of dictyostelium discoideum blocked in expression of all members of the developmentally regulated discoidin multigene family

Mutant strains of D. discoideum are described that can complete morphogenesis and cytodifferentiation but which express vastly reduced levels of the galactose-binding lectins discoidin I and II (less than 1% and 1%-2% respectively) compared to the wild-type control. Mutant cells proceeding through development lack lectin activity, lectin protein, and specific lectin mRNA. In contrast, the genes encoding these proteins are present in their wild-type configurations in the genome. Since these proteins are encoded by four to five discrete genes, the mutations in these strains are most likely in genes involved in the regulation of the expression of members of this multigene family. The results also indicate that the discoidin lectins may not be required for fruiting body construction in this organism. Finally, coupled with the recent ability to transform D. discoideum, these mutants open the way to identification and isolation of regulatory genes and their products.

Antibodies to Synthetic Peptide Immunogens as Probes for Virus Protein Expression and Function

In general, the direction of research into the genetic structure of viruses has been that of tracing phenotype to genotype. The currently available recombinant DNA and nucleic acid sequencing techniques have somewhat reversed this process and we are beginning to find ourselves with genotypes in search of phenotypes, especially as research efforts move from questions about known viruses and viral proteins to questions about recently isolated or little-studied viruses. Indeed, from the nucleotide sequence of a virus, one can learn a great deal about the genetic structure of the viral genome, its coding capacity and its evolutionary history. What is needed, however, is a way to parlay the wealth of genetic information available in the nucleotide sequence into biological experiments. Furthermore, since proteins are the agents responsible for most of the biologically important activities and properties of viruses, one is faced with the not-so-trivial task of identifying the protein products of the viral genes and generating probes to study protein expression, processing, and biological activities. In other words, how does one identify a protein whose existence is merely inferred from the sequence of a string of nucleotides?

Anti-peptide Antibodies: Some Practical Considerations

The observation that antibodies to almost any region of a protein can be induced by a short peptide corresponding to that region has led to the development of a general technology for producing protein-reactive antibodies as well as generating novel insights into the structure of proteins in solution.