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The importance of the human microbiome in health may be the single most valuable development in our conception of the microbial world since Pasteur’s germ theory of the 1860s. Its implications for our understanding of health and pathogenesis are profound. Coupled with the revolution in diagnostics that we are now witnessing – a revolution that changes medicine from a science of symptoms to a science of causes – we cannot continue to develop antibiotics as we have for the past 80 years. Instead, we need to usher in a new conception of the role of antibiotics in treatment: away from single molecules that target broad phylogenetic spectra and towards targeted molecules that cripple the pathogen while leaving the rest of the microbiome largely intact.

Pathogenic bacteria resistant to many or all antibiotics already exist. With the decline in microbiological research at pharmaceutical companies, the high rate at which resistance has evolved and spread has demanded a novel approach to addressing this critical human health issue. In the present paper, we propose a new paradigm in antibiotic discovery and development, one that applies ecological and evolutionary theory to design antimicrobial drugs that are more difficult and/or more costly to resist. In essence, we propose to simply adopt the strategies invented and applied by bacteria for hundreds of millions of years. Our research focuses on bacteriocins, powerful biological weapons, and their use as alternative therapeutics in human health.

The rise of antibiotic-resistant pathogens focuses our attention on the source of antibiotic resistance genes, on the existence of these genes in environments exposed to little or no antibiotics, and on the relationship between resistance genes found in the clinic and those encountered in non-clinical settings. Here, we address the evolutionary history of a class of resistance genes, the SHV β-lactamases. We focus on bla SHV genes isolated both from clinical and non-clinical sources and show that clinically important resistance determinants arise repeatedly from within a diverse pool of bla SHV genes present in the environment. While our results argue against the notion of a single common origin for all clinically derived bla SHV genes, we detect a characteristic selective signature shaping this protein in clinical environments. This clinical signature reveals the joint action of purifying and positive selection on specific residues, including those known to confer extended-spectrum activity. Surprisingly, antibiotic resistance genes isolated from non-clinical—and presumably antibiotic-free—settings also experience the joint action of purifying and positive selection. The picture that emerges undercuts the notion of a separate reservoir of antibiotic resistance genes confined only to clinical settings. Instead, we argue for the presence of a single extensive and variable pool of antibiotic resistance genes present in the environment.

This unit presents a modification of PCR, called anchored PCR, that allows amplification of full-length mRNA when only a small amount of sequence information lying within the mRNA is available. Both the original and reamplifications use an oligo(dT) primer complementary either to the poly(A) tail of the mature mRNA [when amplifying downstream (3') to the known sequence] or to an enzymatically synthesized homopolymer tail added to the cDNA following first strand synthesis [when amplifying upstream (5') to the known sequence]. The two rounds of PCR amplification result in a single product that can be sequenced directly or cloned into an appropriate vector for further analysis.

If genes have been assembled from exon subunits, the frequency with which exons are reused leads to an estimate of the size of the underlying exon universe. An exon database was constructed from available protein sequences, and homologous exons were identified on the basis of amino acid identity; statistically significant matches were determined by Monte Carlo methods. It is estimated that only 1000 to 7000 exons were needed to construct all proteins.

Of the many proteins that are known to be involved in neuronal signaling, one family of gene products, collectively referred to as the G protein-coupled receptors (GPCRs), has received considerable attention. Within the transmembrane domains of GPCRs are clusters of amino acids that tend to be conserved among receptors that bind related ligands. Polymerase chain reaction (PCR)-based approaches to cloning novel GPCRs typically begin with the identification of these well-conserved amino acid motifs, which are then back-translated into degenerate oligonucleotide primers. These pools of degenerate oligonucleotides are the most important variables in PCR cloning of GPCRs. Although GPCRs are used as the focus of this unit, the strategies and techniques described are applicable to the cloning of a wide variety of neuronal gene products. In the first procedure in this unit, either total or poly(A)(+) purified RNA is reverse transcribed into first-strand cDNA. In subsequent steps the cDNA product serves as the template for synthesis and amplification of target receptor sequences by PCR primed with degenerate oligodeoxynucleotides. The product is ready to be cloned and screened as described. Guidelines for database searching are provided to help identify the cloned gene from the known sequence. Typically, only a portion of the receptor coding region is cloned by the above approach. Rapid amplification of cDNA ends (RACE) or anchored PCR is described in this unit and is used to obtain a full-length cDNA amenable for expression studies.

DNA polymorphism in the Y chromosome, examined at a 729-base pair intron located immediately upstream of the ZFY zinc-finger exon, revealed no sequence variation in a worldwide sample of 38 human males. This finding cannot be explained by global constraint on the intron sequence, because interspecific comparisons with other nonhuman primates revealed phylogenetically informative sequence changes. The invariance likely results from either a recent selective sweep, a recent origin for modern Homo sapiens, recurrent male population bottlenecks, or historically small effective male population sizes. A coalescence model predicts an expected time to a most recent common ancestral male lineage of 270,000 years (95 percent confidence limits: 0 to 800,000 years).

We report a rapid technique, based on the polymerase chain reaction (PCR), for the direct targeting, enhancement, and sequencing of previously uncharacterized cDNAs. This method is not limited to previously sequenced transcripts, since it requires only two adjacent or partially overlapping specific primers from only one side of the region to be amplified. These primers can be located anywhere within the message. The specific primers are used in conjunction with nonspecific primers targeted either to the poly(A)+ region of the message or to an enzymatically synthesized d(A) tail. Pairwise combinations of specific and general primers allow for the amplification of regions both 3' and 5' to the point of entry into the message. The amplified PCR products can be cloned, sequenced directly by genomic sequencing, or labeled for sequencing by amplifying with a radioactive primer. We illustrate the power of this approach by deriving the cDNA sequences for the skeletal muscle alpha-tropomyosins of European common frog (Rana temporaria) and zebrafish (Brachydanio rerio) using only 300 ng of a total poly(A)+ preparation. In these examples, we gained initial entry into the tropomyosin messages by using heterologous primers (to conserved regions) derived from the rat skeletal muscle alpha-tropomyosin sequence. The frog and zebrafish sequences are used in an analysis of tropomyosin evolution across the vertebrate phylogenetic spectrum. The results underscore the conservative nature of the tropomyosin molecule and support the notion of a constrained heptapeptide unit as the fundamental structural motif of tropomyosin.