Evidence for biased gene conversion in concerted evolution of ribosomal DNA.

Concerted evolution is the production and maintenance of homogeneity within repeated families of DNA. Two mechanisms--unequal crossing over and biased gene conversion--have been the principal explanations of concerted evolution. Concerted evolution of ribosomal DNA (rDNA) arrays is thought to be largely the result of unequal crossing over. However, concerted evolution of rDNA in parthenogenetic lizards of hybrid origin is strongly biased toward one of two parental sequences, which is consistent with biased gene conversion as the operative mechanism. The apparent gene conversions are independent of initial genome dosage and result in homogenization of rDNA arrays across all nucleolar organizer regions.

Production of goats by somatic cell nuclear transfer.

In this study, we demonstrate the production of transgenic goats by nuclear transfer of fetal somatic cells. Donor karyoplasts were obtained from a primary fetal somatic cell line derived from a 40-day transgenic female fetus produced by artificial insemination of a nontransgenic adult female with semen from a transgenic male. Live offspring were produced with two nuclear transfer procedures. In one protocol, oocytes at the arrested metaphase II stage were enucleated, electrofused with donor somatic cells, and simultaneously activated. In the second protocol, activated in vivo oocytes were enucleated at the telophase II stage, electrofused with donor somatic cells, and simultaneously activated a second time to induce genome reactivation. Three healthy identical female offspring were born. Genotypic analyses confirmed that all cloned offspring were derived from the donor cell line. Analysis of the milk of one of the transgenic cloned animals showed high-level production of human antithrombin III, similar to the parental transgenic line.

Sequences of the primate epsilon-globin gene: implications for systematics of the marmosets and other New World primates.

Sequences of the epsilon-globin gene were determined for five species of marmosets, along with approximately 2 kb of 5' flanking sequence. An analysis of these data, compared with those of other primates strongly supports the classification of Callithrix jacchus and C. geoffroyi into the jacchus group, and C. argentata and C. mauesi into the argentata group. The pygmy marmoset, formerly identified as Cebuella pygmaea joined strongly to the argentata group, indicating that without the pygmy marmoset the genus Callithrix would be paraphyletic. Our data support recent studies which indicate that C. pygmaea should be included in the genus Callithrix. Relationships among other primates were as indicated by previous studies of epsilon-globin sequences. Divergence times were estimated according to a local molecular clock. These calculations indicated the divergence of C. mauesi and C. argentata to be approximately 1.6-1.9 Myr (million years ago), and the most recent common ancestor of the marmosets to be between 4.5 and 4.7 Myr. The latter estimate corresponds well to the date of 4.6 Myr calculated from an independent data set.

Two related G protein-coupled receptors: the distribution of GPR7 in rat brain and the absence of GPR8 in rodents.

GPR7 and GPR8, orphan G protein-coupled receptor (GPCR) genes, expressed in the brain and periphery share highest sequence identity to each other and significant similarity with opioid and somatostatin receptors. To further our knowledge of GPR7's physiological function, we performed in situ hybridization analyses of rat brain to reveal specific patterns of expression in the brain. GPR7 mRNA was found to be discretely localized in areas of the amygdala, hippocampus, hypothalamus and cortex. We previously reported that GPR7 was highly conserved in both human and rodent orthologs while GPR8 was not found in the rodent [9]. We speculated that GPR8 originated after the divergence of the human and rodent. Using primers designed from human GPR8, we isolated lemur GPR8 and subsequently aligned human, monkey, and lemur GPR8 orthologs to design primers recognizing highly conserved regions of GPR8. Using these primers, orthologs of GPR7 and GPR8 were isolated by the PCR from rabbit, tree shrew, and flying lemur, as well as GPR7 in the rat. Subsequent analysis of the clones obtained demonstrated that both GPR7 and GPR8 sequences were highly conserved amongst the species studied, but a rodent GPR8 was not isolated. The absence of a GPR8 gene in the rodent suggests that GPR8 originated from gene duplication of GPR7 after the rodent line diverged from the rabbit, tree shrew, flying lemur, lemur, monkey and human lines. In addition, the taxonomic distribution of GPR8 is consistent with molecular studies grouping rabbits with primates, tree shrews and flying lemurs rather than with rodents.

Resectional gastric bypass is a new alternative in morbid obesity.

BACKGROUND: Severe obesity is a common serious health problem in the United States. Medical therapy is often ineffective. A variety of surgical procedures have been employed for treatment of morbid obesity. Surgical therapy continues to evolve. METHODS: Eighty-five patients have undergone subtotal gastrectomy and retrocolic Roux-en-Y gastrojejunostomy for weight control at our institution. We refer to this procedure as resectional gastric bypass (RGB). Thirty-eight patients have undergone RGB as conversion from failed or problematic prior bariatric procedures. Forty-seven patients have had RGB as their primary bariatric procedure. RESULTS: Twenty-six patients undergoing RGB for conversion of an anatomically or functionally failed prior bariatric procedure have had mean additional weight loss of 37% excess body weight (EBWL) in 18 months follow-up. Twelve patients undergoing RGB for intractable side effects of prior bariatric procedures have all had clinical improvement. Forty-seven patients undergoing RGB as a primary procedure have had EBWL of 53%, in mean follow-up of 11 months. For the entire series, major complications were one anastomotic leak, one reexploration for suspected subphrenic abscess, and one major pulmonary embolus. These patients recovered. There was no mortality in the series. CONCLUSIONS: Resectional gastric bypass is a new alternative for salvage of a failed or problematic prior bariatric procedure. It is also effective as a primary weight control operation.

Assessment of anxiety sensitivity in young American Indians and Alaska Natives.

In the present study, the Anxiety Sensitivity Index [ASI; Behav. Res. Ther. 24 (1986) 1] was administered to 282 American Indian and Alaska Native college students in a preliminary effort to: (a) evaluate the factor structure and internal consistency of the ASI in a sample of Native Americans; (b) examine whether this group would report greater levels of anxiety sensitivity and gender and age-matched college students from the majority (Caucasian) culture lesser such levels; and (c) explore whether gender differences in anxiety sensitivity dimensions varied by cultural group (Native American vs. Caucasian). Consistent with existing research, results of this investigation indicated that, among Native peoples, the ASI and its subscales had high levels of internal consistency, and a factor structure consisting of three lower-order factors (i.e. Physical, Psychological, and Social Concerns) that all loaded on a single higher-order (global Anxiety Sensitivity) factor. We also found that these Native American college students reported significantly greater overall ASI scores as well as greater levels of Psychological and Social Concerns relative to counterparts from the majority (Caucasian) culture. There were no significant differences detected for ASI physical threat concerns. In regard to gender, we found significant differences between males and females in terms of total and Physical Threat ASI scores, with females reporting greater levels, and males lesser levels, of overall anxiety sensitivity and greater fear of physical sensations; no significant differences emerged between genders for the ASI Psychological and Social Concerns dimensions. These gender differences did not vary by cultural group, indicating they were evident among Caucasian and Native Americans alike. We discuss the results of this investigation in relation to the assessment of anxiety sensitivity in American Indians and Alaska Natives, and offer directions for future research with the ASI in Native peoples.

Trauma experience comparison of army forward surgical team surgeons at Ben Taub Hospital and Madigan Army Medical Center(2)(2).

Far forward life-saving surgical care is the mission of an army forward surgical team (FST). Trauma skill maintenance is necessary to complete that mission. A new program has been developed for FST training using the resources of a Level 1 trauma center. We sought to compare the experience of FST surgeons at a major urban trauma center with the yearly trauma experience at an army Level 2 trauma center.General surgeons of the 250th FST prospectively tabulated data for trauma patients during a September 1999 unit deployment to Ben Taub Hospital (Houston, Texas). Data collected included nature and location of injury, hospital admission, and surgical intervention. During 1999, similar data were collected at Madigan Army Medical Center (MAMC) (Ft. Lewis, Washington), home station of the 250th and Level 2 trauma center since November 1998.The FST general surgeons observed 319 injuries. Of those injured, 104 were admitted and 19 underwent urgent operation. Direct participation by FST general surgeons in the operative procedures varied. In 1999, MAMC general surgeons treated 455 trauma victims in direct supervision of Army general surgery residents. Madigan Army Medical Center general surgeons admitted 304 and urgently operated on 57 trauma patients, while 107 patients were transferred to another institution for definitive management of orthopedic and nonoperative neurosurgical injuries.CONCLUSIONS:The volume of trauma surgical cases at MAMC during 1999 was 3 times that seen in the 1-month rotation at Ben Taub. General surgeons performed more trauma and abdominal surgery at MAMC with significantly more direct involvement in patient care and operative procedures. The experience of the 250th FST does not justify trauma sustainment deployments for surgeons from military trauma centers.

Organization and chromosomal location of repetitive DNA sequences in three species of squamate reptiles.

Repetitive DNA sequences were isolated from the genomes of species representing three major clades of squamate reptiles. A repetitive sequence (Cn4C7) was isolated from the New Mexican whiptail lizard, Cnemidophorus neomexicanus. This sequence is distributed throughout the chromosomes, but is more concentrated in the telomeric region. Cn4C7 also hybridizes to the chromosomes of other Cnemidophorus. Some evidence was found for concerted evolution of this repeat in hybrid unisexual lineages. In the lesser earless lizard, Holbrookia maculata, the predominant repeat in the genome is represented by a sequence (Hm1E11) which is restricted to the area flanking the centromere in all species of Holbrookia. Two families of repetitive sequences (one dispersed, and the other telomeric) were isolated from the western diamondback rattlesnake, Crotalus atrox. The type and distribution of repetitive sequences in squamates is often taxon-specific, and may be useful as characters for elucidating taxonomic relationships.

c-myc gene sequences and the phylogeny of bats and other eutherian mammals.

The complete protein-coding sequences of the c-myc proto-oncogene were determined for five species of four new orders of eutherian (placental) mammals. These newly obtained sequences were aligned to each other and to other available orthologs for the phylogenetic estimation of eutherian interordinal relationships. Several measures of sequence difference and base composition were first calculated to assess the major evolutionary properties of the three codon positions and two protein-coding exons of the gene. On the basis of these calculations, different parsimony, distance, and maximum likelihood approaches were adopted, with the most sophisticated involving the separate, then combined, likelihood analyses of the third codon positions of exon 2 versus all other sites. These phylogenetic approaches provided clear support for the grouping of Chiroptera (bats) with Artiodactyla (ruminants, camels, and pigs) and Carnivora (cats, dogs, and their allies), an interordinal arrangement that receives strong corroboration from other lines of evidence including complete mitochondrial DNA sequences. In contrast, these analyses failed to provide strong to reasonable support for any other interordinal group. This study concludes with specific recommendations about sampling and other strategies for maximizing the phylogenetic contributions of the c-myc gene to the continued resolution of the eutherian ordinal tree.

Role of nitrogen assimilation in seed development of soybean.

A nondestructive acetylene reduction assay for nitrogenase activity of soybean (Glycine max L. Merr) field plots is presented. Plots consisted of 120 x 150 x 30 centimeter boxes containing 65 plants. The plants were grown in a medium grade sand under controlled nutrient, moisture, and root temperature conditions. Acetylene at a concentration of 10 milliliters per liter was circulated through manifolds in the chambers; equilibration required 5 minutes, and activity was linear with time. Optimum growth and assay environments resulted in activity of 70 micromoles ethylene per plant per hour. Plant development and yield were comparable to soil-grown companion plots.The well accepted hypothesis that developing seeds deprive the nodules of carbohydrate was not substantiated. The nondestructive acetylene reduction profile did not decline until 30 days after the onset of seed development (R-5). This result was consistent with reports from the literature which indicated that 60% of seasonal nitrogen was fixed after R-5. Further, a high correlation shown between integrated seasonal acetylene reduction and yield (r = 0.999) suggested a cooperative relationship between the roots and shoot. A reduction in source:sink ratio (60% defoliation) after R-5 had no effect on acetylene reduction. This showed that neither an increase in sink demand by the pods nor a carbon shortage during podfill decreased dinitrogen fixation. A conceptual model relating seed growth with carbon and nitrogen assimilation is proposed.

Differences in demographics and risk factors among men attending public v non-public STD clinics in Baltimore, Maryland.

OBJECTIVE: To compare the demographics and risk factors of men who utilise the services of a municipal public sexually transmitted disease (STD) clinic with those who utilise the services provided by a non-public men's STD clinic operated by a not for profit primary care clinic. METHODS: A record based review of the characteristics and STD prevalence of men who visited a non-public STD clinic in Baltimore, Maryland, compared with those of a random sample of male attendees of a public STD clinic. Data abstracted from the records included information on age, race/ethnicity, self reported risk behaviours, and STD tests and results. We used chi2 analysis as well as bivariate and multivariate modelling to compare differences in categorical factors between clinics groups. RESULTS: Men who utilised the services at the non-public STD clinic were more often white (71% v 3%, p<0.001), MSM (65% v 2%, p<0.001), and presented for general screening (52% v 15%, p<0.001) compared to those at the public clinic. In addition, they more frequently reported > or =3 partners (22% v 11%, p=0.005), and having an HIV positive partner (10% v 3%, p=0.005). Factors independently associated with attendance at non-public clinic in multivariate analysis were general screening as reason for visit (OR = 11.0, p<0.001), having 3+ partners in past month (OR=10.5, p=0.002), and "sometimes" using condoms (OR=3.6, p=0.033). CONCLUSIONS: Non-public STD clinics can reach a distinct segment of the male population with high risk sexual behaviours that might not attend public STD clinics.

Evidence on mammalian phylogeny from sequences of exon 28 of the von Willebrand factor gene.

Phylogenetic relationships among 27 extant mammalian species (representing 15 placental orders) were studied using sequences of exon 28 of the gene encoding von Willebrand Factor (vWF), a glycoprotein which functions in blood clotting. Analysis of sequences coding for vWF revealed evidence for several subordinal and superordinal groupings, but the earliest branching sequence of placental mammals was left largely unresolved. Strong support was found for a monophyletic clade consisting of elephants, sea cows, hyraxes, aardvarks, and elephant shrews. This systematic placement of the elephant shrews agrees strongly with two other molecular data sets (interphotoreceptor retinoid binding protein and alpha-lens crystallins) and is consistent with analysis of fossil elephant shrews recently discovered in north Africa. Evidence from vWF sequences agrees with a number of previous molecular and morphological studies in providing strong support for the monophyly of both bats and rodents. The orders Primates, Proboscidea, Carnivora, Perissodactyla, and Artiodactyla were represented by more than one species which joined in each case to form a monophyletic order.

Molecular systematics of the family Mormoopidae (Chiroptera) based on cytochrome b and recombination activating gene 2 sequences.

We examined 1140 bp of the mitochondrial cytochrome b gene and 1398 bp of the nuclear RAG2 gene to investigate the systematics of the eight species of bats within the family Mormoopidae. It was concluded that within the genus Pteronotus there were four valid subgenera: Phyllodia, Chilonycteris, Pteronotus, and an undescribed subgenus. Within Pteronotus, P. parnellii either was part of an unresolved tetratomy with the other three subgenera (cytochrome b data) or was basal (RAG2 and combined data). For three species, P. gymnonotus, P. macleayii, and P. quadridens, our sample revealed little geographic variation. In P. davyi and P. parnellii, the magnitude of genetic distance suggests the possibility of two biological species existing within the currently recognized taxa. Within P. personatus, there was substantial geographic variation partitioned in a step-like fashion among our specimens. Neither of the species within the genus Mormoops showed the deep distance nodes present in P. davyi, P. parnellii, and P. personatus. Cytochrome b and RAG2 data indicated that M. megalophylla evolved recently from its common ancestor. Although there was considerable agreement among the branching patterns for the nuclear and mitochondrial genes, both genes failed to provide robust data concerning the evolutionary relationships among the subgenera.

Purification and Properties of 5-Enolpyruvylshikimate-3-Phosphate Synthase from Dark-Grown Seedlings of Sorghum bicolor.

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase (3-phospho-shikimate 1-carboxyvinyltransferase; EC 2.5.1.19) was purified 1300-fold from etiolated shoots of Sorghum bicolor (L.) Moench. Native polyacrylamide gel electrophoresis revealed three barely separated protein bands staining positive for EPSP synthase activity. The native molecular weight was determined to be 51,000. Enzyme activity was found to be sensitive to metal ions and salts. Apparent K(m) values of 7 and 8 micromolar were determined for the substrates shikimate-3-phosphate and phosphoenolpyruvate (PEP), respectively. The herbicide glyphosate was found to inhibit the enzyme competitively with respect to PEP (K(i) = 0.16 micromolar). Characterization studies support the conclusion of a high degree of similarity between EPSP synthase from S. bicolor, a monocot, and the enzyme from dicots. A similarity to bacterial EPSP synthase is also discussed. Three EPSP synthase isozymes (I, II, III) were elucidated in crude homogenates of S. bicolor shoots by high performance liquid chromatography. The major isozymes, II and III, were separated and partially characterized. No significant differences in pH activity profiles and glyphosate sensitivity were found. This report of isozymes of EPSP synthase from S. bicolor is consistent with other reports for shikimate pathway enzymes, including EPSP synthase.

Overproduction of 5-enolpyruvylshikimate-3-phosphate synthase in a glyphosate-tolerant Petunia hybrida cell line.

Analysis of a Petunia hybrida cell culture (MP4-G) resistant to 1 mM glyphosate revealed a 15- to 20-fold increased level of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase in the herbicide-tolerant strain. Immunoblotting and enzyme kinetic measurements established that the increased EPSP synthase activity resulted from overproduction of a herbicide-sensitive form of the enzyme. Homogeneous enzyme preparations were obtained from the herbicide-tolerant cell line by sequential ion-exchange, hydroxyapatite, hydrophobic-interaction, and molecular sieve chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular sieve chromatography established the Petunia enzyme to be a monomeric protein with Mr 49,000-55,800. Km values for phosphoenolpyruvate and shikimate 3-phosphate were about 14 and 18 microM, respectively. Glyphosate inhibited the enzyme competitively with phosphoenolpyruvate (Ki = 0.17 microM). These experiments provide further evidence that EPSP synthase is a major site of glyphosate action in plant cells.

Sequences from the 5' flanking region of the epsilon-globin gene support the relationship of Callicebus with the pitheciins.

The purpose of this study was to determine nucleotide sequences from the 5' flanking region of the epsilon-globin gene of selected platyrrhine primates and to analyze the data for phylogenetic information and estimated times of divergence. We report new sequence data for two species of New World monkeys, Callicebus torquatus and Pithecia irrorata. We analyzed these data in conjunction with homologous sequences from other primate species. The data support the hypothesis that the titi monkeys (Callicebus) and seed predators (Tribe Pitheciini) form a clade (Subfamily Pitheciinae), and also provide limited support for that subfamily being allied with the atelines. We also present estimated dates of divergence for the Callicebus and pitheciin lineages.

Evidence on primate phylogeny from epsilon-globin gene sequences and flanking regions.

Phylogenetic relationships among various primate groups were examined based on sequences of epsilon-globin genes. epsilon-globin genes were sequenced from five species of strepsirhine primates. These sequences were aligned and compared with other known primate epsilon-globin sequences, including data from two additional strepsirhine species, one species of tarsier, 19 species of New World monkeys (representing all extant genera), and five species of catarrhines. In addition, a 2-kb segment upstream of the epsilon-globin gene was sequenced in two of the five strepsirhines examined. This upstream sequence was aligned with five other species of primates for which data are available in this segment. Domestic rabbit and goat were used as outgroups. This analysis supports the monophyly of order Primates but does not support the traditional prosimian grouping of tarsiers, lorisoids, and lemuroids; rather it supports the sister grouping of tarsiers and anthropoids into Haplorhini and the sister grouping of lorisoids and lemuroids into Strepsirhini. The mouse lemur (Microcebus murinus) and dwarf lemur (Cheirogaleus medius) appear to be most closely related to each other, forming a clade with the lemuroids, and are probably not closely related to the lorisoids, as suggested by some morphological studies. Analysis of the epsilon-globin data supports the hypothesis that the aye-aye (Daubentonia madagascariensis) shares a sister-group relationship with other Malagasy strepsirhines (all being classified as lemuroids). Relationships among ceboids agree with findings from a previous epsilon-globin study in which fewer outgroup taxa were employed. Rates of molecular evolution were higher in lorisoids than in lemuroids.

Mammalian evolution and the interphotoreceptor retinoid binding protein (IRBP) gene: convincing evidence for several superordinal clades.

Phylogenetic relationships of 25 mammalian species representing 17 of the 18 eutherian orders were examined using DNA sequences from a 1.2-kb region of the 5' end of exon 1 of the single-copy nuclear gene known as interphotoreceptor retinoid binding protein (IRBP). A wide variety of methods of analysis of the DNA sequence, and of the translated products, all supported a five-order clade consisting of elephant shrew (Macroscelidea)/aardvark (Tubulidentata)/and the paenungulates (hyracoids, sirenians, and elephants), with bootstrap support in all cases of 100%. The Paenungulata was also strongly supported by these IRBP data. In the majority of analyses this monophyletic five-order grouping was the first branch off the tree after the Edentata. These results are highly congruent with two other recent sources of molecular data. Another superordinal grouping, with similar 100% bootstrap support in all of the same wide-ranging types of analyses, was Artiodactyla/Cetacea. Other superordinal affinities, suggested by the analyses, but with less convincing support, included a Perissodactyla/Artiodactyla/Cetacea clade, an Insectivora/Chiroptera clade, and Glires (an association of rodents and lagomorphs).

1 -Antitrypsin deficiency and neonatal hepatitis.

Five out of 28 infants investigated in a regional survey of neonatal hepatitis were found to have genetically-determined deficiency of alpha(1)-antitrypsin (ZZ phenotype). The clinical course and pathological changes varied considerably. All five infants had an acute hepatitis-like illness, and although this subsided cirrhosis later developed in three cases. The remaining two infants had minimal abnormalities of the liver function tests at 12 and 18 months of age, and one had increased hepatic fibrosis. Australia antigen was found in the serum of three infants, and Australia antigen or antibody in one or both parents of these and of one further case whose serum was negative. It is suggested that the association of neonatal hepatitis with alpha(1)-antitrypsin deficiency may be commoner than previously realized and that Australia antigen acts as a trigger factor in these cases.