New records of early Paleocene (earliest Torrejonian) plesiadapiforms from northeastern Montana, USA, provide a window into the diversification of stem primates.

Plesiadapiforms (putative stem primates) appear in the fossil record shortly after the Cretaceous/Paleogene boundary and subsequently radiated throughout the Paleocene into a taxonomically and ecomorphologically diverse group. The oldest known plesiadapiforms come from early Puercan (the oldest North American Land Mammal 'age' [NALMA] of the Cenozoic) deposits in northeastern Montana, and all records of Puercan plesiadapiforms are taxonomically restricted to members of the Purgatoriidae and the enigmatic genus Pandemonium. Plesiadapiform diversity substantially increased in the following Torrejonian NALMA, but the sparse record of faunas between the Puercan and the well-known middle and late Torrejonian has hampered our understanding of this important interval in early primate evolution. Here we report new plesiadapiform dental fossils from early Torrejonian (To1) deposits from the Tullock Member of the Fort Union Formation in northeastern Montana that record several poorly known taxa including members of the Purgatoriidae, Paromomyidae and Pandemonium, and that document the largest and most diverse assemblage of To1 plesiadapiforms known. We describe a new species of the purgatoriid Ursolestes (Ursolestes blissorum, sp. nov.) that represents the largest plesiadapiform known from the early Paleocene and, among other taxa, provides additional evidence that the temporal range of purgatoriids extended into the Torrejonian. Large sample sizes of the oldest known paromomyid, Paromomys farrandi, allowed us to document intraspecific variability and one undescribed tooth locus. Our observations illuminate changes in dental morphology of some taxa that occurred in To1 and may inform the acquisition of certain diagnostic plesiadapiform dental characters. We evaluate plesiadapiform species richness, mean body mass and body-mass disparity through the Paleocene and reveal unrecognized levels of richness in To1 and a general trend of stable body mass and body-mass disparity. Our findings contribute to documented patterns of plesiadapiform provincialism in the early Paleocene and shed light on the early stages of their Torrejonian radiation.

Earliest Palaeocene purgatoriids and the initial radiation of stem primates.

Plesiadapiform mammals, as stem primates, are key to understanding the evolutionary and ecological origins of Pan-Primates and Euarchonta. The Purgatoriidae, as the geologically oldest and most primitive known plesiadapiforms and one of the oldest known placental groups, are also central to the evolutionary radiation of placentals and the Cretaceous-Palaeogene biotic recovery on land. Here, we report new dental fossils of Purgatorius from early Palaeocene (early Puercan) age deposits in northeastern Montana that represent the earliest dated occurrences of plesiadapiforms. We constrain the age of these earliest purgatoriids to magnetochron C29R and most likely to within 105-139 thousand years post-K/Pg boundary. Given the occurrence of at least two species, Purgatorius janisae and a new species, at the locality, we provide the strongest support to date that purgatoriids and, by extension, Pan-Primates, Euarchonta and Placentalia probably originated by the Late Cretaceous. Within 1 million years of their arrival in northeastern Montana, plesiadapiforms outstripped archaic ungulates in numerical abundance and dominated the arboreal omnivore-frugivore niche in mammalian local faunas.

Isolation and characterization of Staphylococcus aureus strains from a Paso del Norte dairy.

The primary purpose of this study was to determine if methicillin-resistant Staphylococcus aureus (MRSA) strains could be identified in the milk of dairy cattle in a Paso del Norte region dairy of the United States. Using physiological and PCR-based identification schemes, a total of 40 Staph. aureus strains were isolated from 29 raw milk samples of 133 total samples analyzed. Pulsed-field gel electrophoresis after digestion with the SmaI enzyme revealed that the 40 confirmed strains were represented by 5 pulsed-field types, which each contained 3 or more strains. Of 7 hospital strains isolated from cows undergoing antibiotic therapy, 3 demonstrated resistance to 3 or more antimicrobial classes and displayed similar pulsed-field gel electrophoresis patterns. A secondary purpose of this study was to elucidate the evolutionary relationships of strains isolated in this study to genomically characterized Staph. aureus strains. Therefore, Roche 454 GS (Roche Diagnostics Corp., Dallas, TX) pyrosequencing was used to produce draft genome sequences of an MRSA raw milk isolate (H29) and a methicillin-susceptible Staph. aureus (PB32). Analysis using the BLASTn database (http://blast.ncbi.nlm.nih.gov/) demonstrated that the H29 draft genome was highly homologous to the human MRSA strain JH1, yet the ß-lactamase plasmid carried by H29 was different from that carried by JH1. Genomic analysis of H29 also clearly explained the multidrug resistance phenotype of this raw milk isolate. Analysis of the PB32 draft genome (using BLASTn) demonstrated that this raw milk isolate was most related to human MRSA strain 04-02981. Although PB32 is not a MRSA, the PB32 draft genome did reveal the presence of a unique staphylococcal cassette mec (SCCmec) remnant. In addition, the PB32 draft genome revealed the presence of a novel bovine staphylococcal pathogenicity island, SaPIbovPB32. This study demonstrates the presence of clones closely related to human and (or) bovine Staph. aureus strains circulating in a dairy herd.

A search for Drosophila neural precursor genes identifies ran.

The Drosophila ran gene has been isolated in a differential cDNA screen designed to identify genes that are dynamically expressed in embryonic neuroblasts. The guanine triphosphate (GTP)-binding Ran protein, a member of the Ras superfamily, has been shown to participate in a variety of transport related processes in other organisms. Drosophila ran codes for a 216 amino acid (aa) protein that shares 78% and 86% identity with the yeast and human Ran proteins, respectively. Database searches have identified a second Drosophila ran gene, ran-like. The predicted Ran-like protein shares 59% identity with its isoform. Embryo in situ mRNA localization of ran and ran-like expression reveals that both are maternally expressed; however zygotic ran expression is restricted to central nervous system (CNS) neuroblasts undergoing late lineage formation, while ran-like expression is detected in the developing trachea and salivary gland. To investigate the significance of ran-restricted CNS expression, we have targeted its misexpression to different temporal windows of CNS development. In addition, a dominant-negative mutant form of ran was targeted to the developing CNS and to the larval eye/antenna imaginal disc to assess the role of ran-dependent functions. Embryonic CNS misexpression of the mutant, but not wild-type, ran results in larval death. Neither wild-type nor mutant ran misexpression had any detectable effect on embryonic CNS lineage specification, nuclear transport of a number of CNS-specific transcription factors or axonal guidance. However, expression of the dominant-negative mutant ran in the developing eye/antenna disc did result in a severe adult eye phenotype marked by apoptosis of photoreceptor, cone and pigment cells.

Nerfin-1 and -2, novel Drosophila Zn-finger transcription factor genes expressed in the developing nervous system.

To gain insight into the regulatory networks controlling Drosophila neural-identity decisions, we have identified new neuronal precursor genes by performing an in situ hybridization screen of differentially selected embryonic head cDNAs. Here, we describe the molecular characteristics and expression profile of nerfin-1, a novel pan-neural precursor gene. This paper also documents the embryonic expression of another structurally related gene, nerfin-2. During early CNS development, nerfin-1 gene expression is activated in neuroblasts (NBs) prior to lineage formation. However, after early sublineage development, nerfin-1 expression shifts from NBs to ganglion mother cells (GMCs) but is not expressed in neurons or glia. Differing from nerfin-1, nerfin-2 is expressed only in a subset of brain neurons. Possessing a conserved putative DNA-binding domain, the predicted Nerfin-1 and -2 proteins belong to a subfamily of Zn-finger transcription factors with cognates identified in nematode, mouse and man.

Programmed transformations in neuroblast gene expression during Drosophila CNS lineage development.

During Drosophila embryonic CNS development, the sequential neuroblast (NB) expression of four proteins, Hunchback (Hb), Pou-homeodomain proteins 1 and 2 (referred to collectively as Pdm), and Castor (Cas), identifies a transcription factor network regulating the temporal development of all ganglia. The Zn-finger proteins Hb and Cas, acting as repressors, confine Pdm expression to a narrow intermediate temporal window; this results in the generation of three panneural domains whose cellular constituents are marked by expression of Hb, Pdm, or Cas (R. Kambadur et al., 1998, Genes Dev. 12, 246-260). Seeking to identify the cellular mechanisms that generate these expression compartments, we studied the lineage development of isolated NBs in culture. We found that the Hb, Pdm, and Cas expression domains are generated by transitions in NB gene expression that are followed by gene product perdurance within sequentially produced sublineages. Our results also indicate that following Cas expression, many CNS NBs continue their asymmetric divisions generating additional progeny, which can be identified by the expression of the bHLH transcription factor Grainyhead (Gh). Gh appears to be a terminal embryonic CNS lineage marker. Taken together, these studies indicate that once NBs initiate lineage development, no additional signaling between NBs and the neuroectoderm and/or mesoderm is required to trigger the temporal progression of Hb --> Pdm --> Cas --> Gh expression during NB outgrowth.

Comparative genomics of the eukaryotes.

A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.

Characterization of Alu repeats that are associated with trinucleotide and tetranucleotide repeat microsatellites.

The association of subclasses of Alu repetitive elements with various classes of trinucleotide and tetranucleotide microsatellites was characterized as a first step toward advancing our understanding of the evolution of microsatellite repeats. In addition, information regarding the association of specific classes of microsatellites with families of Alu elements was used to facilitate the development of genetic markers. Sequences containing Alu repeats were eliminated because unique primers could not be designed. Various classes of microsatellites are associated with different classes of Alu repeats. Very abundant and poly(A)-rich microsatellite classes (ATA, AATA) are frequently associated with an evolutionarily older subclass of Alu repeats, AluSx, whereas most of GATA and CA microsatellites are associated with a recent Alu subfamily, AluY. Our observations support all three possible mechanisms for the association of Alu repeats to microsatellites. Primers designed using a set of sequences from a particular microsatellite class showed higher homology with more sequences of that class than probes designed for other classes. We developed an efficient method of prescreening GGAA and ATA microsatellite clones for Alu repeats with probes designed in this study. We also showed that Alu probes labeled in a single reaction (multiplex labeling) could be used efficiently for prescreening of GGAA clones. Sequencing of these prescreened GGAA microsatellites revealed only 5% Alu repeats. Prescreening with primers designed for ATA microsatellite class resulted in the reduction of the loss of markers from approximately 50% to 10%. The new Alu probes that were designed have also proved to be useful in Alu-Alu fingerprinting.

Multistep denaturation and hierarchy of disulfide bond cleavage of a monoclonal antibody.

A humanized, monoclonal antibody of the IgG4 class was treated with SDS at room temperature. Analysis using SDS-PAGE revealed the progressive formation of a series of denaturation intermediates, of increasing apparent molecular weights. The detectable species migrated with apparent molecular weights of 137, 152, 162, 182, 196, and 210 kDa. Antibody not incubated in SDS had an apparent molecular weight of 137 kDa, while boiling in SDS provoked the immediate conversion of all antibody to the slowest migrating form (210 kDa). A study designed to test if all rungs of the ladder were equally cleavable by mild treatment with mercaptoethanol revealed small amounts of a novel form, and conditions were devised to generate large amounts of this novel form. The novel form, before full denaturation, migrated at the position of unheated, intact antibody (137 kDa), while full denaturation in SDS converted the novel form to a species migrating at about 190 kDa. The novel form, before full denaturation, was identified as HHL ***L, where the asterisks indicate noncovalent binding of one light chain to HHL. This noncovalent complex remains intact during SDS-PAGE. The novel form, after full denaturation, was identified as HHL. This study, with earlier studies, reveals that different pathways of reductive cleavage are taken by different antibodies, and that the most sensitive disulfide bond is different for different antibodies. Our results on antibody denaturation serve as a warning to those who use two-dimensional gels for the analysis of antibodies.

Identification of the murine beige gene by YAC complementation and positional cloning.

The beige mutation is a murine autosomal recessive disorder, resulting in hypopigmentation, bleeding and immune cell dysfunction. The gene defective in beige is thought to be a homologue of the gene for the human disorder Chediak-Higashi syndrome. We have identified the murine beige gene by in vitro complementation and positional cloning, and confirmed its identification by defining mutations in two independent mutant alleles. The sequence of the beige gene message shows strong nucleotide homology to multiple human ESTs, one or more of which may be associated with the Chediak-Higashi syndrome gene. The amino acid sequence of the Beige protein revealed a novel protein with significant amino acid homology to orphan proteins identified in Saccharomyces cerevisiae, Caenorhabditis elegans and humans.

Identification and characterization of the mouse obesity gene tubby: a member of a novel gene family.

The mutated gene responsible for the tubby obesity phenotype has been identified by positional cloning. A single base change within a splice donor site results in the incorrect retention of a single intron in the mature tub mRNA transcript. The consequence of this mutation is the substitution of the carboxy-terminal 44 amino acids with 24 intron-encoded amino acids. The normal transcript appears to be abundantly expressed in the hypothalamus, a region of the brain involved in body weight regulation. Variation in the relative abundance of alternative splice products is observed between inbred mouse strains and appears to correlate with an intron length polymorphism. This allele of tub is a candidate for a previously reported diet-induced obesity quantitative trait locus on mouse chromosome 7.

Chromosomal assignment of 2900 tri- and tetranucleotide repeat markers using NIGMS somatic cell hybrid panel 2.

Two thousand nine hundred and thirty-one tri- and tetranucleotide short tandem repeat polymorphisms (STRPs) developed by the Cooperative Human Linkage Center were assigned to chromosomes using the NIGMS somatic cell hybrid mapping panel 2 and an efficient pooling strategy. Approximately 82% of all STRPs tested were assigned by this method, with 96.7% accuracy. Many of the single chromosome cell lines contained portions of additional chromosomes, confirming previous reports. The cell lines for chromosomes 6, 14, and 20 contained extensive portions of other chromosomes. Five previously unreported chromosomal contaminants were identified and are reported. A new pooling strategy was designed to minimize ambiguous assignments.

Development of a screening set for new (CAG/CTG)n dynamic mutations.

The expansion of a (CAG/CTG)n triplet repeat has been found to be associated with at least seven genetic diseases, suggesting that this mechanism of disease may be fairly common. To accelerate the discovery of new loci containing (CAG/CTG)n triplet expansions, we have isolated numerous genomic clones containing this class of repeats. We have developed 338 sequence-tagged sites (STSs) containing (CAG/CTG)n repeat sequences. Two hundred ninety-nine STSs were unambiguously assigned to chromosomes, and 89 of the total were assigned to YACs. The 141 STSs that were developed based on (CAG/CTG)n repeats of at least seven units were genotyped on four reference CEPH individuals to estimate their polymorphic quality.

Survey of trinucleotide repeats in the human genome: assessment of their utility as genetic markers.

Genetic markers based upon PCR amplification of short tandem repeat-containing sequence tagged sites (STSs) have become the standard for genetic mapping. We have completed a survey based on the direct isolation of representative members of each of the 10 trinucleotide repeat classes to determine their relative abundance, repeat size distribution, and general utility as genetic markers. Trinucleotide repeats, depending on the repeat class, are one to two orders of magnitude less frequent than (AC)n repeats. The average size of trinucleotide repeats sequenced was less than 15 repeat units in length, and only three of the STSs developed for this study demonstrated more than 25 repeats units. The (AAT)n class of repeats are the most abundant and also the most frequently polymorphic. Other classes of trinucleotide repeat classes observed to be frequently polymorphic include (AAC)n, (ACT)n, (ATC)n and (AAG)n; however, the relative abundance of these classes is less than that observed for the (AAT)n class of repeats. Based upon this initial survey, we have initiated saturation cloning of the (AAT)n class of repeats. At the time of submission of this manuscript, we have developed, as part of the Cooperative Human Linkage Center (CHLC), more than 415 new high heterozygosity (AAT)n genetic markers (more than two alleles in four individuals) and 200 new low heterozygosity (AAT)n STSs from this larger screening effort combined with the initial survey.

Chromosomal localization and cDNA cloning of the genes (DDB1 and DDB2) for the p127 and p48 subunits of a human damage-specific DNA binding protein.

DDB is a damage-specific DNA binding protein whose binding activity is absent from a minority of cell strains from individuals with xeroderma pigmentosum Group E, a human hereditary disease characterized by defective nucleotide excision DNA repair and an increased incidence of skin cancer. The binding activity from HeLa cells is associated with polypeptides of M(r) 124,000 and 41,000 as determined by SDS-polyacrylamide gels. This report describes the isolation of full-length human cDNAs encoding each polypeptide of DDB. The predicted peptide molecular masses based on open reading frames are 127,000 and 48,000. When expressed in an in vitro rabbit reticulocyte system, the p48 subunit migrates with an M(r) of 41 kDa on SDS-polyacrylamide gels, similarly to the peptide purified from HeLa cells. There is no significant homology between the derived p48 peptide sequence and any proteins in current databases, and the derived peptide sequence of p127 has homology only with the monkey DDB p127 (98% nucleotide identity and only one conserved amino acid substitution). Using a fluorescence in situ hybridization technique, the DDB p127 locus (DDB1) was assigned to the chromosomal location 11q12-q13, and the DDB p48 locus (DDB2) to 11p11-p12.

Correction of the DNA repair defect in xeroderma pigmentosum group E by injection of a DNA damage-binding protein.

Cells from a subset of patients with the DNA-repair-defective disease xeroderma pigmentosum complementation group E (XP-E) are known to lack a DNA damage-binding (DDB) activity. Purified human DDB protein was injected into XP-E cells to test whether the DNA-repair defect in these cells is caused by a defect in DDB activity. Injected DDB protein stimulated DNA repair to normal levels in those strains that lack the DDB activity but did not stimulate repair in cells from other xeroderma pigmentosum groups or in XP-E cells that contain the activity. These results provide direct evidence that defective DDB activity causes the repair defect in a subset of XP-E patients, which in turn establishes a role for this activity in nucleotide-excision repair in vivo.

The fluoroquinolones.

At the Eastern Section Meeting of the Triological Society January 26, 1990, Levinson et al reported outstanding success with ciprofloxacin in the treatment of malignant otitis externa. Moreover, several of the individuals so managed had been refractory to conventional therapy with semisynthetic penicillin and aminoglycoside protocols. This new class of antibiotics may result in a profound change in our management of patients with otologic, neurotologic, and skull base infections.