Identification and Cloning of Genes Involved in Specific Desulfurization of Dibenzothiophene by Rhodococcus sp. Strain IGTS8

The gram-positive bacterium Rhodococcus sp. strain IGTS8 is able to remove sulfur from certain aromatic compounds without breaking carbon-carbon bonds. In particular, sulfur is removed from dibenzothiophene (DBT) to give the final product, 2-hydroxybiphenyl. A genomic library of IGTS8 was constructed in the cosmid vector pLAFR5, but no desulfurization phenotype was imparted to Escherichia coli. Therefore, IGTS8 was mutagenized, and a new strain (UV1) was selected that had lost the ability to desulfurize DBT. The genomic library was transferred into UV1, and several colonies that had regained the desulfurization phenotype were isolated, though free plasmid could not be isolated. Instead, vector DNA had integrated into either the chromosome or a large resident plasmid. DNA on either side of the inserted vector sequences was cloned and used to probe the original genomic library in E. coli. This procedure identified individual cosmid clones that, when electroporated into strain UV1, restored desulfurization. When the origin of replication from a Rhodococcus plasmid was inserted, the efficiency with which these clones transformed UV1 increased 20- to 50-fold and they could be retrieved as free plasmids. Restriction mapping and subcloning indicated that the desulfurization genes reside on a 4.0-kb DNA fragment. Finally, the phenotype was transferred to Rhodococcus fascians D188-5, a species normally incapable of desulfurizing DBT. The mutant strain, UV1, and R. fascians produced 2-hydroxybiphenyl from DBT when they contained appropriate clones, indicating that the genes for the entire pathway have been isolated.     

Prevalence of nine mutations among Jewish and non-Jewish Gaucher disease patients.

The frequency of nine different mutated alleles known to occur in the glucocerebrosidase gene was determined in 247 Gaucher patients, of whom 176 were of Jewish extraction, 2 were Jewish with one converted parent, and 69 were of non-Jewish origin. DNA was prepared from peripheral blood, active glucocerebrosidase sequences were amplified by using the PCR technique, and the mutations were identified by using the allele-specific oligonucleotide hybridization method. The N37OS mutation appeared in 69.77% of the mutated alleles in Jewish patients and in 22.86% of the mutated alleles in non-Jews. The 84GG mutation, which has not been found so far among non-Jewish patients, existed in 10.17% of the disease alleles among Jewish patients. The IVS + 1 mutation constituted 2.26% of the disease alleles among Jewish patients and 1.43% among the non-Jewish patients. RecTL, a complex allele containing four single-base-pair changes, occurred in 2.26% of the alleles in Jewish patients and was found in two (1.43%) of the patients of non-Jewish extraction. Another complex allele, designated "RecNciI" and containing three single-point mutations, appeared in 7.8% of alleles of non-Jewish patients and in only two (0.56%) of the Jewish families. The prevalence of the L444P mutation among non-Jewish Gaucher patients was 31.43%, while its prevalence among Jewish patients was only 4.24%. The prevalence of two other point mutations--D409H and R463C--was 5.00% and 3.57%, respectively, among non-Jewish patients and was not found among the Jewish Gaucher patient population. The prevalence of the R496H mutation, found so far only among Jewish patients, was 1.13%. The results presented demonstrate that seven mutations identify 90.40% of the mutations among Jewish patients and that these seven mutations allow diagnosis of only 73.52% of the non-Jewish patients. Identification of additional mutant alleles will enhance the accuracy of carrier detection.     

Founder effect in a Belgian-Dutch fragile X population

For many years, the high prevalence of the fragile X syndrome was thought to be caused by a high mutation frequency. The recent isolation of the FMR1 gene and identification of the most prevalent mutation enable a more precise study of the fragile X mutation. As the vast majority of fragile X patients show amplification of an unstable trinucleotide repeat, DNA studies can now trace back the origin of the fragile X mutation. To date, de novo mutations leading to amplification of the CGG repeat have not yet been detected. Recently, linkage disequilibrium was found in the Australian and US populations between the fragile X mutation and adjacent polymorphic markers, suggesting a founder effect of the fragile X mutation. We present here a molecular study of Belgian and Dutch fragile X families. No de novo mutations could be found in 54 of these families. Moreover, we found significant (P < 0.0001) linkage disequilibrium in 68 unrelated fragile X patients between the fragile X mutation and an adjacent polymorphic microsatellite at DXS548. This suggests that a founder effect of the fragile X mutation also exists in the Belgian and Dutch populations. Both the absence of new mutations and the presence of linkage disequilibrium suggest that a few ancestral mutations are responsible for most of the patients with fragile X syndrome.     

Loss of functionally important and regionally endemic species from streams forced into intermittency by global warming

Climate change is altering hydrological cycles globally, and in Mediterranean (med-) climate regions it is causing the drying of river flow regimes, including the loss of perennial flows. Water regime exerts a strong influence over stream assemblages, which have developed over geological timeframes with the extant flow regime. Consequently, sudden drying in formerly perennial streams is expected to have large, negative impacts on stream fauna. We compared contemporary (2016/17) macroinvertebrate assemblages of formerly perennial streams that became intermittently flowing (since the early 2000s) to assemblages recorded in the same streams by a study conducted pre-drying (1981/82) in the med-climate region of southwestern Australia (the Wungong Brook catchment, SWA), using a multiple before-after, control-impact design. Assemblage composition in the stream reaches that remained perennial changed very little between the studies. In contrast, recent intermittency had a profound effect on species composition in streams impacted by drying, including the extirpation of nearly all Gondwanan relictual insect species. New species arriving at intermittent streams tended to be widespread, resilient species including desert-adapted taxa. Intermittent streams also had distinct species assemblages, due in part to differences in their hydroperiods, allowing the establishment of distinct winter and summer assemblages in streams with longer-lived pools. The remaining perennial stream is the only refuge for ancient Gondwanan relict species and the only place in the Wungong Brook catchment where many of these species still persist. The fauna of SWA upland streams is becoming homogenised with that of the wider Western Australian landscape, as drought-tolerant, widespread species replace local endemics. Flow regime drying caused large, in situ alterations to stream assemblage composition and demonstrates the threat posed to relictual stream faunas in regions where climates are drying.     

The structure and evolution of the human salivary proline-rich protein gene family

We present the nucleotide sequences of four members of the six-member human salivary prolinerich protein (PRP) gene family. The four genes are PRB1 and PRB2, which encode basic PRPs, and PRB3 and PRB4, which encode glycosylated PRPs. Each PRB gene is approximately 4.0 kb in length and contains four exons, the third of which is entirely composed of 63-bp tandem repeats and encodes the proline-rich portion of the protein products. Exon 3 contains different numbers of tandem repeats in the different PRB genes. Variation in the numbers of these repeats is also responsible for length variations in different alleles of the PRB genes. We have determined a probable evolutionary history of the human PRP gene family by comparing the nucleotide sequences of the six PRP genes. The present-day six PRP loci probably evolved from a single ancestral gene by four sequential gene duplications, leading to six genes that fall into three subsets, each consisting of two genes. During this evolutionary process, multiple rearrangements and gene conversion occurred mainly in the region from the 3 end of IVS2 and the 3 end of exon 3.     

DIVERGENT AMBULATORY AND GROOMING BEHAVIOR IN SERIALLY BOTTLENECKED LINES OF THE HOUSEFLY

The extent of genome-wide restructuring predicted in bottleneck models of speciation is addressed in assays of non-reproductive behavior in lines of the housefly. After five serial founder-flush cycles of one of three sizes (1, 4, or 16 pairs), each bottleneck line showed significant differentiation from the outbred control in ambulatory levels and grooming sequences in videotaped records of precopulatory activity. Only one line (4-pair) showed overall lethargy which was associated to inbreeding depression in egg-to-adult viability, thus exemplifying a case of probable extinction due to bottlenecks. The two most hyperactive lines (1- and 16-pair) showed very similar directions of differentiation from the control in locomotor activity and grooming behavior, as well as in mating behavior evaluated from a separate study. This high congruence suggested that directional selection toward the phenotypic optima of the ancestor operated on the bottleneck populations and that a 10-fold difference in theoretical inbreeding coefficients did not affect the magnitude of response. The remaining two bottleneck lines showed some independence from these general trajectories, their divergence along minor axes of ancestral intercorrelation structure possibly being more important to the formation of new species. Significant perturbations of the thresholds for execution of grooming and locomotor movements suggested increased evolutionary potential for ritualization (i.e., sexual selection for adoption of non-reproductive behavior into courtship repertoire) due to bottlenecks.     

AN ANALYSIS OF SELECTIONAL RESPONSE IN RELATION TO A POPULATION BOTTLENECK

Selection for increased morphometric shape (ratio of wing length to thorax width) was compared between control (nonbottlenecked) populations and bottlenecked populations founded with two male–female pairs of flies. Contrary to neutral expectation, selectional response was not reduced in bottlenecked populations, and the mean realized heritabilities and additive genetic variances were higher for the bottlenecked lines than for the nonbottlenecked lines. Additive genetic variances based on these realized heritabilities were consistent with independent estimates of genetic variances based on parent–offspring covariances. Joint scaling tests applied to the crosses between selected lines and their controls revealed significant nonadditive components of genetic variance in the ancestor, which were not detected in the crosses involving bottlenecked lines. The nonbottlenecked lines responded principally by changes in one trait or the other (wing length or thorax width) but not in both, and regardless of which trait responded, larger trait size was dominant and epistatic to smaller size. Stabilizing selection for morphometric shape in the ancestor likely molded the genetic architecture to include nonadditive genetic effects.     

Identification of a Novel Stage of Ribosome/Nascent Chain Association with the Endoplasmic Reticulum Membrane

Protein translocation in the mammalian endoplasmic reticulum (ER) occurs cotranslationally and requires the binding of translationally active ribosomes to components of the ER membrane. Three candidate ribosome receptors, p180, p34, and Sec61p, have been identified in binding studies with inactive ribosomes, suggesting that ribosome binding is mediated through a receptor-ligand interaction. To determine if the binding of nascent chain-bearing ribosomes is regulated in a manner similar to inactive ribosomes, we have investigated the ribosome/nascent chain binding event that accompanies targeting. In agreement with previous reports, indicating that Sec61p displays the majority of the ER ribosome binding activity, we observed that Sec61p is shielded from proteolytic digestion by native, bound ribosomes. The binding of active, nascent chain bearing ribosomes to the ER membrane is, however, insensitive to the ribosome occupancy state of Sec61p. To determine if additional, Sec61p independent, stages of the ribosome binding reaction could be identified, ribosome/nascent chain binding was assayed as a function of RM concentration. At limiting RM concentrations, a protease resistant ribosome-membrane junction was formed, yet the nascent chain was salt extractable and cross-linked to Sec61p with low efficiency. At nonlimiting RM concentrations, bound nascent chains were protease and salt resistant and cross-linked to Sec61p with higher efficiency. On the basis of these and other data, we propose that ribosome binding to the ER membrane is a multi-stage process comprised of an initial, Sec61p independent binding event, which precedes association of the ribosome/nascent chain complex with Sec61p.     

Insulin receptors in 3T3 fibroblasts : Relationship to growth phase, transformation and differentiation into new cell types

The amount of [¹²⁵I]insulin binding per 2 × 10⁶ cells is measured in three lines of mouse embryonic 3T3 fibroblast at different growth stages. Insulin binding is found to be lowest in growing cells of all three types, increasing as cells reach stationary phase. Binding in 3T3-M cells approaches a plateau as cells become stationary. Insulin binding in 3T3-L cells, many of which differentiate into adipocytes following cessation of growth, show further increase in insulin binding post-confluence, in parallel with their differentiation into adipocytes. Binding of insulin in spontaneously transformed cells is higher at all phases of growth than the other two lines, rising to a much higher eventual plateau at approx. 17 days post-confluence. Scatchard plots of insulin binding tend to reflect the same degree of relative insulin binding in these three cell lines. Previously starved cells of all three types exhibit a drop in insulin binding following their first feeding, which corresponds with a second growth spurt in response to nutrients in fresh serum. These results suggest that insulin, as reflected by binding per cell, may play only a minor role in actively growing adequately fed cells of all three types, its major role developing as these cells approach confluence. It is also suggested that higher insulin binding in transformed vs non-transformed cells may indicate a special role for insulin in the loss of contact inhibition, by preserving transport of limiting nutrients in dense, nutrient-depleted transformed cultures.     

Sexual preferences of male hamsters (Mesocricetus auratus) for conspecifics in different endocrine conditions

The behavioral responses of sexually experienced male hamsters toward a pair of anesthetized conspecifics were investigated. Males spent significantly more time licking, sniffing, and mounting neonatally and adult castrated males than intact males. Adult castrated males receiving oil injections were preferred over castrates receiving exogenous testosterone propionate (TP). Ovariectomized females were preferred over intact males, adult castrated males, or spayed females receiving exogenous TP. It was concluded that the absence of an androgen-dependent factor(s) renders an animal more sexually attractive.