Genomic organization of human arylamine N-acetyltransferase Type I reveals alternative promoters that generate different 5'-UTR splice variants with altered translational activities

In humans, a polymorphic gene encodes the drug-metabolizing enzyme NATI (arylamine N-acetyltransferase Type 1), which is widely expressed throughout the body. While the protein-coding region of NATI is contained within a single exon, examination of the human EST (expressed sequence tag) database at the NCBI revealed the presence of nine separate exons, eight of which were located in the 5'non-coding region of NATI. Differential splicing produced at least eight unique mRNA isoforms that could be grouped according to the location of the first exon, which suggested that NATI expression occurs from three alternative promoters. Using RT (reverse transcriptase)-PCR, we identified one major transcript in various epithelial cells derived from different tissues. In contrast, multiple transcripts were observed in blood-derived cell lines (CEM, THP-1 and Jurkat), with a novel variant, not identified in the EST database, found in CEM cells only. The major splice variant increased gene expression 9-11-fold in a luciferase reporter assay, while the other isoforrns were similar or slightly greater than the control. We examined the upstream region of the most active splice variant in a promoter-reporter assay, and isolated a 257 bp sequence that produced maximal promoter activity. This sequence lacked a TATA box, but contained a consensus Sp1 site and a CAAT box, as well as several other putative transcription-factor-binding sites. Cell-specific expression of the different NATI transcripts may contribute to the variation in NATI activity in vivo.

Isolation of polymorphic tetranucleotide microsatellite for the large-billed scrubwren (Sericomis magnirostris)

We isolated 12 polymorphic microsatellite markers for the large-billed scrubwren Sericornis magnirostris from genomic libraries enriched for (AAGG)(n) and (AACC)(n) repetitive elements and characterized them in 11 individuals. The number of alleles ranged from four to 15 per locus with the observed heterozygosity ranging from 0.14 to 0.91. These markers will be useful to address questions concerning population genetic structure and models of speciation.

Isolation of polymorphic tetranucleotide microsatellite markers for the grey-headed robin (Poecilodryas albispecularis)

We isolated 12 polymorphic microsatellite markers for the grey-headed robin Poecilodryas albispecularis from genomic libraries enriched for (AAGG)n and (AACC)n repetitive elements and characterized them in 12 individuals. The number of alleles ranges from three to nine per locus with the observed heterozygosity ranging from 0.33 to 0.90. These markers will be useful for analysis of questions concerning population genetic structure and testing models of speciation.

Functional characterization and genomic organization of the human Na+-sulfate cotransporter hNaS2 gene (SLC13A4)

Sulfate plays an essential role in human growth and development. Here, we characterized the functional properties of the human Na+-sulfate cotransporter (hNaS2), determined its tissue distribution, and identified its gene (SLC13A4) structure. Expression of hNaS2 protein in Xenopus oocytes led to a Na+-dependent transport of sulfate that was inhibited by thiosulfate, phosphate, molybdate. selenate and tungstate, but not by oxalate, citrate, succinate, phenol red or DIDS. Transport kinetics of hNaS2 determined a K, for sulfate of 0.38 mM, suggestive of a high affinity sulfate transporter. Na+ kinetics determined a Hill coefficient of 1.6 +/- 0.6, suggesting a Na: SO42- stoichiometry of 2:1. hNaS2 mRNA was highly expressed in placenta and testis, with intermediate levels in brain and lower levels found in the heart, thymus, and liver. The SLC13A4 gene contains 16 exons, spanning over 47 kb in length. Its 5'-flanking region contains CAAT- and GC-box motifs, and a number of putative transcription factor binding sites, including GATA-1, AP-1, and AP-2 consensus sequences. This is the first study to characterize hNaS2 transport kinetics, define its tissue distribution, and resolve its gene (SLC13A4) structure and 5' flanking region. (C) 2004 Elsevier Inc. All rights reserved.

Chloroplast and total genomic diversity in the endemic Costa Rican tree Lonchocarpus costaricensis (J.D. Smith) Pittier (Papilionaceae).

In Mesoamerica, tropical dry forest is a highly threatened habitat, and species endemic to this environment are under extreme pressure. The tree species, Lonchocarpus costaricensis is endemic to the dry northwest of Costa Rica and southwest Nicaragua. It is a locally important species but, as land has been cleared for agriculture, populations have experienced considerable reduction and fragmentation. To assess current levels and distribution of genetic diversity in the species, a combination of chloroplast-specific (cpDNA) and whole genome DNA markers (amplified fragment length polymorphism, AFLP) were used to fingerprint 121 individual trees in 6 populations. Two cpDNA haplotypes were identified, distributed among populations such that populations at the extremes of the distribution showed lowest diversity. A large number (487) of AFLP markers were obtained and indicated that diversity levels were highest in the two coastal populations (Cobano, Matapalo, H = 0.23, 0.28 respectively). Population differentiation was low overall, F-ST = 0.12, although Matapalo was strongly differentiated from all other populations (F-ST = 0.16-0.22), apart from Cobano (F., = 0.11). Spatial genetic structure was present in both datasets at different scales: cpDNA was structured at a range-wide distribution scale, whilst AFLP data revealed genetic neighbourhoods on a population scale. In general, the habitat degradation of recent times appears not to have yet impacted diversity levels in mature populations. However, although no data on seed or saplings were collected, it seems likely that reproductive mechanisms in the species will have been affected by land clearance. It is recommended that efforts should be made to conserve the extant genetic resource base and further research undertaken to investigate diversity levels in the progeny generation.

Optimization of a transplant model to assess skin reconstitution from stem cell-enriched primary human keratinocyte populations

Given that an important functional attribute of stem cells in vivo is their ability to sustain tissue regeneration, we set out to establish a simple and easy technique to assess this property from candidate populations of human keratinocyte stem cells in an in vivo setting. Keratinocytes were inoculated into devitalized rat tracheas and transplanted subcutaneously into SCID mice, and the epithelial lining regenerated characterized to establish the validity of this heterotypic model. Furthermore, the rate and quality of epidermal tissue reconstitution obtained from freshly isolated unfractionated vs. keratinocyte stem cell-enriched populations was tested as a function of (a) cell numbers inoculated; and (b) the inclusion of irradiated support keratinocytes and dermal cells. Rapid and sustained epidermal tissue regeneration from small numbers of freshly isolated human keratinocyte stem cells validates the utilization of this simple and reliable model system to assay for enrichment of epidermal tissue-reconstituting cells.

Analysis of the Pasteurella multocida outer membrane sub-proteome and its response to the in vivo environment of the natural host

This study describes the identification of outer membrane proteins (OMPs) of the bacterial pathogen Pasteurella multocida and an analysis of how the expression of these proteins changes during infection of the natural host. We analysed the sarcosine-insoluble membrane fractions, which are highly enriched for OMPs, from bacteria grown under a range of conditions. Initially, the OMP-containing fractions were resolved by 2-DE and the proteins identified by MALDI-TOF MS. In addition, the OMP-containing fractions were separated by 1-D SDS-PAGE and protein identifications were made using nano LC MS/MS. Using these two methods a total of 35 proteins was identified from samples obtained from organisms grown in rich culture medium. Six of the proteins were identified only by 2-DE MALDI-TOF MS, whilst 17 proteins were identified only by 1-D LC MS/MS. We then analysed the OMPs from P. multocida which had been isolated from the bloodstream of infected chickens (a natural host) or grown in iron-depleted medium. Three proteins were found to be significantly up-regulated during growth in vivo and one of these (Pm0803) was also up-regulated during growth in iron-depleted medium. After bioinformatic analysis of the protein matches, it was predicted that over one third of the combined OMPs predicted by the bioinformatics sub-cellular localisation tools PSORTB and Proteome Analyst, had been identified during this study. This is the first comprehensive proteomic analysis of the P. multocida outer membrane and the first proteomic analysis of how a bacterial pathogen modifies its outer membrane proteome during infection.

Arf6-independent GPI-anchored protein-enriched early endosomal compartments fuse with sorting endosomes via a Rab5/phosphatidylinositol-3 '-kinase-dependent machinery

In the process of internalization of molecules from the extracellular milieu, a cell uses multiple endocytic pathways, consequently generating different endocytic vesicles. These primary endocytic vesicles are targeted to specific destinations inside the cell. Here, we show that GPI-anchored proteins are internalized by an Arf6-independent mechanism into GPI-anchored protein-enriched early endosomal compartments (GEECs). Internalized GPI-anchored proteins and the fluid phase are first visualized in GEECs that are acidic, primary endocytic structures, negative for early endosomal markers, Rab4, Rab5, and early endosome antigen (EEA)1. They subsequently acquire Rab5 and EEA1 before homotypic fusion with other GEECs, and heterotypic fusion with endosomes containing cargo from the clathrin-dependent endocytic pathway. Although, the formation of GEECs is unaffected by inhibition of Rab5 GTPase and phosphatidylinositol-3'-kinase (PI3K) activity, their fusion with sorting endosomes is dependent on both activities. Overexpression of Rab5 reverts PI3K inhibition of fusion, providing evidence that Rab5 effectors play important roles in heterotypic fusion between the dynamin-independent GEECs and clathrin- and dynamin-dependent sorting endosomes.

Construction and analysis of a metagenomic library from an enhanced biological phosphorus removal biomass

The enhanced biological phosphorus removal (EBPR) process is regularly used for the treatment of wastewater, but suffers from erratic performance. Successful EBPR relies on the growth of bacteria called polyphosphate-accumulating organisms (PAOs), which store phosphorus intracellularly as polyphosphate, thus removing it from wastewater. Metabolic models have been proposed which describe the measured chemical transformations, however genetic evidence is lacking to confirm these hypotheses. The aim of this research was to generate a metagenomic library from biomass enriched in PAOs as determined by phenotypic data and fluorescence in situ hybridisation (FISH) using probes specific for the only described PAO to date, Candidatus Accumulibacter phosphatis. DNA extraction methods were optimised and two fosmid libraries were constructed which contained 93 million base pairs of metagenomic data. Initial screening of the library for 16S rRNA genes revealed fosmids originating from a range of non-pure-cultured wastewater bacteria. The metagenomic libraries constructed will provide the ability to link phylogenetic and metabolic data for bacteria involved in nutrient removal from wastewater. Keywords DNA extraction; EBPR; metagenomic library; 16S rRNA gene.

Effect of free ammonia and free nitrous acid concentration on the anabolic and catabolic processes of an enriched Nitrosomonas culture

The effects of free ammonia (FA; NH3) and free nitrous acid (FNA; HNO2) concentrations on the metabolisms of an enriched ammonia oxidizing bacteria (AOB) culture were investigated using a method allowing the decoupling of growth and energy generation processes. A lab-scale sequencing batch reactor (SBR) was operated for the enrichment of an AOB culture. Fluorescent in-situ hybridization (FISH) analysis showed that 82% of the bacterial population in the SBR bound to the NEU probe specifically designed for Nitrosomonas europaea. Batch tests were carried out to measure the oxygen and ammonium consumption rates by the culture at various FA and FNA levels, in the presence or absence of inorganic carbon (CO2, HCO3, and CO32-). It was revealed that FA of up to 16.0 mgNH(3)-N (.) L-1, which was the highest concentration used in this study, did not have any inhibitory effect on either the catabolic or anabolic processes of the Nitrosomonas culture. In contrast, FNA inhibited both the growth and energy production capabilities of the Nitrosomonas culture. The inhibition on growth initiated at approximately 0.10 mgHNO(2)-(NL-1)-L-., and the data suggested that the biosynthesis was completely stopped at an FNA concentration of 0.40 mgHNO(2)-N (.) L-1. The inhibition on energy generation initiated at a slightly lower level but the Nitrosomonas culture was still oxidizing ammonia at half of the maximum rate at an FNA concentration of 0.50-0.63 mgHNO(2)-N (.) L-1. The affinity constant of the Nitrosomonas culture with respect to ammonia was determined to be 0.36 mgNH3-N (.) L-1, independent of the presence or absence of inorganic carbon. (c) 2006 Wiley Periodicals, Inc.

Isolation of polymorphic tetranucleotide microsatellite markers for the green-eyed tree frog (Litoria genimaculata)

We have isolated 16 polymorphic microsatellite markers for the green-eyed tree frog, Litoria genimaculata, from genomic libraries enriched for (AAGG)(n) and (AAAG)(n) repetitive elements. The number of alleles ranges from four to 14 per locus with the observed heterozygosity ranging from 0.36 to 1.00. These markers will be useful for analysis of questions concerning population genetic structure and speciation.

Isolation of polymorphic tetranucleotide microsatellite markers for the ornate nursery frog Cophixalus ornatus

We have isolated 18 polymorphic microsatellite loci for Cophixalus ornatus from genomic libraries enriched for (AAAG)(n), (AACC)(n) and (AAGG)(n) repetitive elements. The number of alleles ranges from five to 22 per locus with the observed heterozygosity ranging from 0.10 to 0.92. These markers will be useful for the analysis of population structure in C. ornatus and testing alternative models of speciation.

The inhibitory effects of free nitrous acid on the energy generation and growth processes of an enriched Nitrobacter culture

The inhibitory effects of nitrite (NO2-)/free nitrous acid (HNO2-FNA) on the metabolism of Nitrobacter were investigated using a method allowing the decoupling of the growth and energy generation processes. A lab-scale sequencing batch reactor was operated for the enrichment of a Nitrobacter culture. Fluorescent in situ hybridization (FISH) analysis showed that 73% of the bacterial population was Nitrobacter. Batch tests were carried out to assess the oxygen and nitrite consumption rates of the enriched culture at low and high nitrite levels, in the presence or absence of inorganic carbon. It was observed that in the absence of CO2, the Nitrobacter culture was able to oxidize nitrite at a rate that is 76% of that in the presence of CO2, with an oxygen consumption rate that is 85% of that measured in the presence of CO2. This enabled the impacts of nitrite/FNA on the catabolic and anabolic processes of Nitrobacter to be assessed separately. FNA rather than nitrite was likely the actual inhibitor to the Nitrobacter metabolism. It was revealed that FNA of up to 0.05 mg HNO2-N center dot L-1 (3.4 mu M), which was the highest FNA concentration used in this study, did not have any inhibitory effect on the catabolic processes of Nitrobacter. However, FNA initiated its inhibition to the anabolic processes of Nitrobacter at approximately 0.011 mg HNO2-N center dot L-1 (0.8 mu M), and completely stopped biomass synthesis at a concentration of approximately 0.023 mg HNO2-N center dot L-1 (1.6 mu M). The inhibitory effect could be described by an empirical inhibitory model proposed in this paper, but the underlying mechanisms remain to be revealed.

Microarray-based comparative genomic hybridisation of breast cancer patients receiving neoadjuvant chemotherapy

We analysed the molecular genetic profiles of breast cancer samples before and after neoadjuvant chemotherapy with combination doxorubicin and cyclophosphamide (AC). DNA was obtained from microdissected frozen breast core biopsies from 44 patients before chemotherapy. Additional samples were obtained before the second course of chemotherapy (D21) and after the completion of the treatment (surgical specimens) in 17 and 21 patients, respectively. Microarray-based comparative genome hybridisation was performed using a platform containing approx5800 bacterial artificial chromosome clones (genome-wide resolution: 0.9 Mb). Analysis of the 44 pretreatment biopsies revealed that losses of 4p, 4q, 5q, 12q13.11–12q13.12, 17p11.2 and 17q11.2; and gains of 1p, 2p, 7q, 9p, 11q, 19p and 19q were significantly associated with oestrogen receptor negativity. 16q21–q22.1 losses were associated with lobular and 8q24 gains with ductal types. Losses of 5q33.3–q4 and 18p11.31 and gains of 6p25.1–p25.2 and Xp11.4 were associated with HER2 amplification. No correlations between DNA copy number changes and clinical response to AC were found. Microarray-based comparative genome hybridisation analysis of matched pretreatment and D21 biopsies failed to identify statistically significant differences, whereas a comparison between matched pretreatment and surgical samples revealed a statistically significant acquired copy number gain on 11p15.2–11p15.5. The modest chemotherapy-driven genomic changes, despite profound loss of cell numbers, suggest that there is little therapeutic selection of resistant non-modal cell lineages.

Lineages of acidophilic archaea revealed by community genomic analysis

Novel, low-abundance microbial species can be easily overlooked in standard polymerase chain reaction (PCR)-based surveys. We used community genomic data obtained without PCR or cultivation to reconstruct DNA fragments bearing unusual 16S ribosomal RNA ( rRNA) and protein-coding genes from organisms belonging to novel archaeal lineages. The organisms are minor components of all biofilms growing in pH 0.5 to 1.5 solutions within the Richmond Mine, California. Probes specific for 16S rRNA showed that the fraction less than 0.45 micrometers in diameter is dominated by these organisms. Transmission electron microscope images revealed that the cells are pleomorphic with unusual folded membrane protrusions and have apparent volumes of < 0.006 cubic micrometer.