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PubMed1984

Different recombinant murine leukemia viruses use different cell surface receptors.

Retroviruses can be grouped by viral interference measurements into classes which use common cell surface receptors. We previously tested a large number of isolates of mink cell focus-inducing (MCF) murine leukemia viruses (MuLVs), and reported that all of them share a distinct receptor on NIH/3T3 cells (A. Rein, Virology 120, 251, 1982). We now extend this generalization to several additional recombinant isolates, including two (SL3-2 and GPA-V2) which would not be considered MCFs on the basis of host-range data. We note the superiority of interference tests, based on positive, unambiguous data, over host-range tests for virus classification. We also show that in contrast to the MCFs, which are all derived from ecotropic MuLVs, a recombinant derived from wild mouse amphotropic MuLV (S. Rasheed et al., Int. J. Cancer 29, 345, 1982) uses a unique receptor on NIH/3T3 cells. This suggests that (a) mouse cells contain more than one type of endogenous env sequence; and (b) there is some specificity in the generation of recombinants, since ecotropic MuLVs appear to give rise only to MCFs, while amphotropic MuLV has generated a distinct type of recombinant. It also represents a second case (in addition to the MCFs) in which an env gene recombinant is more pathogenic than its exogenous parent. We also show that xenotropic MuLV does not interfere with MCFs in NZB mouse cells; thus, despite the close homology between MCF and xenotropic env sequences, the gp70 of xenotropic MuLV appears to have no detectable affinity for the MCF receptor.

Animals↗

PubMed2000

Positional cloning utilizing genomic DNA microarrays: the Niemann-Pick type C gene as a model system.

A major obstacle in positional cloning is identifying the specific mutated gene from within a large physical contig. Here we describe the application of DNA microarray technology to a defined genomic region (physical map) to identify: (i) exons without a priori sequence data and (ii) the disease gene based on differential gene expression in a recessive disorder. The feasibility was tested using resources from the positional cloning of the Neimann-Pick Type C (NP-C) disease gene, NPC1. To identify NPC1 exons and optimize the technology, an array was generated from genomic fragments of the 110-kb bacterial artificial chromosome, 108N2, which encodes NPC1. First, as a test case for blindly identifying exons, fluorescently labeled NPC1 cDNA identified 108N2 fragments that contained NPC1 exons, many of which also contained intronic sequences and could be used to determine part of the NPC1 genomic structure. Second, to demonstrate that the NPC1 disease gene could be identified based upon differential gene expression, subarrays of 108N2 fragments were hybridized with fluorescently labeled cDNA probes generated from total RNA from hamster cell lines differentially expressing NPC1. A probe derived from the NP-C cell line CT60 did not detect NPC1 exons or other genomic fragments from 108N2. In contrast, several NPC1 exons were detected by a probe generated from the non-NP-C cell line 911D5A13, which was derived from CT60, and expressed NPC1 as a consequence of stable transduction with a YAC that contains NPC1 and encompasses 108N2. Thus, the array technology identified NPC1 as a candidate gene based on a physical contig and differential NPC1 expression between NP-C and non-NP-C cells. This technique should facilitate gene identification when a physical contig exists for a region of interest and mutations result in changes in the mRNA level of the disease gene or portions thereof.

Animals↗

PubMed1996

Noncoding RNA for CR20, a cytokinin-repressed gene of cucumber.

The CR20 gene was identified as a cytokinin-repressed gene in excised cotyledons of cucumber. We determined the sequences of some CR20 cDNAs with different structures and sequenced genomic clones for CR20. This gene consisted of three exons, and there were at least three types of transcript, which seemed to be generated by alternative splicing of the second intron. None of the CR20 transcripts included a long open reading frame (ORF). We isolated a cDNA of Arabidopsis thaliana with cucumber CR20 cDNA as a probe. This cDNA for a gene designated AtCR20-1 also lacked a long ORF. A region of 180 nucleotides was conserved in the CR20 RNA of cucumber and the AtCR20-1 RNA of Arabidopsis, although the homology was relatively low when the entire sequences were compared. Each conserved region consisted of seven elements, and seems to form stable secondary structure. These suggest that CR20 RNA may function as an RNA that is not translated into a protein.

Alternative Splicing↗

PubMed2004

Information hidden in signals and macromolecules I. Symbolic time-series analysis.

We describe the conceptual background and practical implementation of some recently developed techniques for the analysis of symbol sequences and symbolic time series. We emphasize their associated software realization, the WinGramm suite of programs, that includes programs for the calculation of conditional entropies, context-free grammatical complexity, algorithmic distance and redundancy, as well as for the generation of surrogates that preserve symbol pairs and triplets. We demonstrate the usefulness of these programs by means of two illustrative examples, taken from computational neuroscience. In the first one, we obtain evidence of the Markovian character of the cortical inter spike intervals of the rat before penicillin treatment, and its disappearance afterwards. In the second one, we extend previous investigations about neural spike-trains generated by the isolated neuron of the slowly adapting stretch receptor organ (SAO), in order to classify sequences of different length of known neural behaviors. We include new spike trains, digitized employing the optimal partition procedure described by Steuer, Molgedey, Ebeling, & Jimenez-Montaño, (2001).

Journal Article↗

PubMed2002

Identification of a novel transcript of X25, the human gene involved in Friedreich ataxia.

Friedreich ataxia (FRDA) is caused by a GAA triplet expansion in the first intron of the X25 gene. The X25 gene encodes a 210-amino acid protein, frataxin (A isoform). Here, we report the identification of a new transcript of the X25 gene generated by alternative splicing by the use of a second donor splice site in the intron 4. Full-length cDNA transcript sequence revealed an insertion of 8 bp between 4 and 5a exon sequence. This event leads to a frameshift in the mRNA reading frame and introduces a new stop codon at position 589. Therefore, this X25 transcript variant may encode a 196-amino acid protein, the A1 isoform, that structurally differs from the main A isoform of 210 amino acids after residue 160. In all human tissues analyzed, reverse transcription-polymerase chain reaction experiments demonstrated that the A1 isoform was expressed at low levels compared with the predominant A isoform. No difference in A and A1 isoform expression rate was detected between FRDA patients and normal controls. We did not find an A1 like splice variant in rodents.

Alternative Splicing↗

PubMed2002

Combining biometric and symbolic models for customized, automated prosthesis design.

In a previous paper [Artif. Intell. Med. 5 (1993) 431] we described RaPiD, a knowledge-based system for designing dental prostheses. The present paper discusses how RaPiD has been extended using techniques from computer vision and logic grammars. The first employs point distribution and active shape models (ASMs) to determine dentition from images of casts of patient's jaws. This enables a design to be customized to, and visualised against, an image of a patient's dentition. The second is based on the notion of a path grammar, a form of logic grammar, to generate a path linking an ordered sequence of subcomponents. The shape of an important and complex prosthesis component can be automatically seeded in this fashion. Combining these models now substantially automates the design process, beginning with a photograph of a dental cast and ending with an annotated and validated design diagram ready to guide manufacture.

Anthropometry↗

PubMed2025

Gain-of-function PPM1D mutations attenuate ischemic stroke.

Identification of genetic aberrations in stroke, the second leading cause of death worldwide, is of paramount importance for understanding the disease pathogenesis and generating new therapies. Whole-genome sequencing from 10,241 ischemic stroke patients identified eight patients carrying gain-of-function mutations on coding variants in the protein phosphatase magnesium-dependent 1 δ (PPM1D) gene. Patients carrying PPM1D mutations exhibit better stroke-related clinical phenotypes, including improvements in peripheral inflammation, fibrinogen, low-density lipoprotein, cholesterol and plateletcrit level. Experimental brain ischemia in Ppm1d-deficient (Ppm1d-/-) mice resulted in enlarged lesions and pronounced neurological impairments. Spatial transcriptomics revealed a distinct Ppm1d-associated gene expression pattern, indicating disrupted endothelial homeostasis during ischemic brain injury. Proteomic analysis demonstrated that differentially expressed proteins in primary brain endothelial cells from Ppm1d-/- mice were significantly enriched in the peroxisome proliferator-activated receptors (PPARs)-mediated metabolic signaling. Mechanistically, Ppm1d deficiency promoted aberrant fatty acid β-oxidation and increased oxidative stress, which impaired endothelial cell function through the PPARα pathway. A small molecule, T2755, was identified to engage Trp427 and stabilize PPM1D, thereby mitigating ischemic brain injury in mice. Collectively, we find that PPM1D protects against ischemic brain injury and validates its pharmacological stabilizer T2755 as a promising therapy for ischemic stroke. Gain-of-function PPM1D mutations attenuate ischemic cerebral injury. Whole-genome sequencing data of 10,241 ischemic stroke patients from the Third Chinese National Stroke Registry (CNSR-III) identified eight patients with gain-of-function mutations in the protein phosphatase magnesium-dependent 1 δ (PPM1D) gene (17q23.2). These mutation carriers displayed improved peripheral inflammation, decreased fibrinogen, low-density lipoprotein, cholesterol and plateletcrit level. Ppm1d-deficient (Ppm1d-/-) mice exhibited exacerbated stroke outcomes, characterized by enlarged infarct volumes, disrupted cerebrovascular architecture, and enhanced neuro-inflammation. Mechanistically, Ppm1d deficiency induced the disturbance of endothelial fatty acid metabolism involving the PPARα pathway. Through integrated computational modeling, virtual screening, and in vitro validation, T2755 was identified as a small molecule PPM1D stabilizer. Pharmacological PPM1D stabilization with T2755 significantly attenuated ischemic brain injury in murine models.

Aged↗

PubMed1998

Functional genomics: probing plant gene function and expression with transposons.

Transposable elements provide a convenient and flexible means to disrupt plant genes, so allowing their function to be assessed. By engineering transposons to carry reporter genes and regulatory signals, the expression of target genes can be monitored and to some extent manipulated. Two strategies for using transposons to assess gene function are outlined here: First, the PCR can be used to identify plants that carry insertions into specific genes from among pools of heavily mutagenized individuals (site-selected transposon mutagenesis). This method requires that high copy transposons be used and that a relatively large number of reactions be performed to identify insertions into genes of interest. Second, a large library of plants, each carrying a unique insertion, can be generated. Each insertion site then can be amplified and sequenced systematically. These two methods have been demonstrated in maize, Arabidopsis, and other plant species, and the relative merits of each are discussed in the context of plant genome research.

Journal Article↗

PubMed2004

Dimerization of the transmembrane domain of Integrin alphaIIb subunit in cell membranes.

Homo- and hetero-oligomeric interactions between the transmembrane (TM) helices of integrin alpha and beta subunits may play an important role in integrin activation and clustering. As a first step to understanding these interactions, we used the TOXCAT assay to measure oligomerization of the wild-type alpha(IIb) TM helix and single-site TM domain mutants. TOXCAT measures the oligomerization of a chimeric protein containing a TM helix in the Escherichia coli inner membrane via the transcriptional activation of the gene for chloramphenicol acetyltransferase. We found the amount of chloramphenicol acetyltransferase induced by the wild-type alpha(IIb) TM helix was approximately half that induced by the strongly dimerizing TM helix of glycophorin A, confirming that the alpha(IIb) TM domain oligomerizes in biological membranes. Mutating each of the alpha(IIb) TM domain residues to either Ala, Leu, Ile, or Val revealed that a GXXXG motif mediates oligomerization. Further, we found that the residue preceding each glycine contributed to the oligomerization interface, as did the residue at position i + 4 after the second Gly of GXXXG. Thus, the sequence XXVGXXGGXXXLXX is critical for oligomerization of alpha(IIb) TM helix. These data were used to generate an atomic model of the alpha(IIb) homodimer, revealing a family of structures with right-handed crossing angles of 40 degrees to 60 degrees, consistent with a 4.0-residue periodicity, and with an interface rotated by 50 degrees relative to glycophorin A. Thus, although the alpha(IIb) TM helix makes use of the GXXXG framework, neighboring residues have evolved to engineer its dimerization interface, enabling it to subserve specific and specialized functions.

Amino Acid Motifs↗

PubMed2006

Second generation subtyping: a proposed PulseNet protocol for multiple-locus variable-number tandem repeat analysis of Shiga toxin-producing Escherichia coli O157 (STEC O157).

Most bacterial genomes contain tandem duplications of short DNA sequences, termed "variable-number tandem repeats" (VNTR). A subtyping method targeting these repeats, multiple-locus VNTR analysis (MLVA), has emerged as a powerful tool for characterization of clonal organisms such as Shiga toxin-producing Escherichia coli O157 (STEC O157). We modified and optimized a recently published MLVA scheme targeting 29 polymorphic VNTR regions of STEC O157 to render it suitable for routine use by public health laboratories that participate in PulseNet, the national and international molecular subtyping network for foodborne disease surveillance. Nine VNTR loci were included in the final protocol. They were amplified in three PCR reactions, after which the PCR products were sized using capillary electrophoresis. Two hundred geographically diverse, sporadic and outbreak- related STEC O157 isolates were characterized by MLVA and the results were compared with data obtained by pulsed-field gel electrophoresis (PFGE) using XbaI macrorestriction of genomic DNA. A total of 139 unique XbaI PFGE patterns and 162 MLVA types were identified. A subset of 100 isolates characterized by both XbaI and BlnI macrorestriction had 62 unique PFGE and MLVA types. Although the clustering of isolates by the two subtyping systems was generally in agreement, some discrepancies were observed. Importantly, MLVA was able to discriminate among some epidemiologically unrelated isolates which were indistinguishable by PFGE. However, among strains from three of the eight outbreaks included in the study, two single locus MLVA variants and one double locus variant were detected among epidemiologically implicated isolates that were indistinguishable by PFGE. Conversely, in three other outbreaks, isolates that were indistinguishable by MLVA displayed multiple PFGE types. An additional more extensive multi-laboratory validation of the MLVA protocol is in progress in order to address critical issues such as establishing epidemiologically relevant interpretation guidelines for the MLVA data.

Base Sequence↗

PubMed1995

Role of the beta-amyloid protein in Alzheimer's disease.

A major histopathological hallmark of Alzheimer's disease (AD) is the presence of amyloid deposits in the parenchyma of the amygdala, hippocampus, and neocortex. The principal component of amyloid is the beta-amyloid protein (A beta), a 39-43 amino acid peptide composed of a portion of the transmembrane domain and the extracellular domain of the amyloid precursor protein (APP). APP occurs as several A beta-containing isoforms of 695, 751, and 770 amino acids, with the latter two APP containing a domain that shares structural and functional homologies with Kunitz serine protease inhibitors. In cultured cells, APP mature through the constitutive secretory pathway, and some cell surface-bound APP are cleaved by an enzyme, designated as alpha-secretase, within the A beta domain, an event that precludes A beta amyloidogenesis. Several studies have delineated two additional pathways of APP processing: first, an endosomal/lysosomal pathway generates a complex set of APP-related membrane-bound fragments, some of which contain the entire A beta sequence; and, second, by mechanisms which are not fully understood, A beta 1-40 is secreted into the conditioned medium in vitro and is present in cerebrospinal fluid in vivo. The intracellular sites of enzymes responsible for proteolytic cleavage at the NH2 and COOH termini of A beta, termed gamma- and beta-secretase, respectively, have not been identified. Finally, recent molecular genetic investigations have identified a variety of mutations in APP that segregate with early-onset familial AD and with hereditary cerebral hemorrhage with amyloid, Dutch type (HCHWA-D). Several of these mutations appear to influence APP processing and result in the production of higher levels or longer A beta-related peptides that are inherently more fibrillogenic. Although a variety of lines of evidence implicates APP/A beta in AD, the mechanisms by which A beta influences the biology and vulnerability of neural cells are not fully understood but are very active areas of investigation. This review focuses on the present state of our understanding of APP and A beta in the context of AD.

Alzheimer Disease↗

PubMed2002

Angiotensin II's antiproliferative effects mediated through AT2-receptors depend on down-regulation of SM-20.

Angiotensin II (ANG II) inhibits proliferation and induces differentiation through AT2 receptors. However, target genes involved in this process are not well characterized. We studied PC12 cells, a rat pheochromocytoma cell line exclusively expressing AT2 receptors. ANG II attenuated proliferation of PC12 cells without concomitant induction of apoptosis. To identify potential novel genes involved in the antimitogenic actions of ANG II, we performed differential display analysis of PC12 cells after challenge with 10(-7) M ANG II for 6 hours. One identified gene selected for further study that was down-regulated by ANG II in PC12 cells was SM-20. This gene has been previously isolated from vascular smooth muscle cells treated with mitogens by differential hybridization. Recent findings show a homology of SM-20 with enzymes involved in the regulation of hypoxia inducible factor 1. ANG II suppressed mRNA expression of SM-20 in PC12 cells after only 30 minutes, as detected by Northern blotting. This effect was antagonized by an AT2 receptor blocker, but not by losartan. A rabbit polyclonal antibody was generated against a peptide sequence of SM-20 and detected a major band of the predicted size of 40 kd and a second 33-kd band that likely represents a processed form present in mitochondria. Immunohistochemistry revealed a granular staining of the cytoplasm of PC12 cells compatible with a previously described mitochondrial localization of SM-20 protein. Western blots confirmed the down-regulation of SM-20 protein in PC12 cells subsequent to incubation with ANG II. SM-20 transcripts were also reduced by ANG II acting on AT2 receptors in rat glomerular endothelial cells that express both AT1 and AT2 receptors. SM-20 antisense, but not sense, phosphothioate-modified oligonucleotides reduced base-line proliferation of PC12 cells. In contrast, inducible overexpression of SM-20 using the ecdysone system prevented the antiproliferative effects of ANG II in PC12 cells. In summary, our study identified SM-20 as an essential component of ANG II's growth-suppressive effects mediated through AT2 receptors. This gene apparently plays an important role in the regulatory processes determining whether a cell should undergo differentiation, apoptosis, or proliferation.

Angiotensin II↗

PubMed1987

5' long terminal repeats of myc-associated proviruses appear structurally intact but are functionally impaired in tumors induced by avian leukosis viruses.

B-cell lymphomas induced in chickens infected with avian leukosis viruses are characterized by integration of the virus within the cellular myc locus and alteration of c-myc expression. Although avian leukosis viruses are intact, replication-competent retroviruses, the structures of many myc-associated proviruses are altered by deletions, raising the possibility that proviral defectiveness plays an essential role in oncogenesis. We found that all myc-associated proviruses in 21 independent tumors had deletions, which were confined to the viral genome and did not extend into adjacent cellular sequences. Deletions were not random but, in at least 85% of the myc-associated proviruses, involved a region near the 5' end of the proviral genome where elements implicated in control of viral gene expression have been localized. A second class of deletions involved sequences in the 3' half of the viral genome and included the splice acceptor site used in generating viral env mRNA. Both the 5' and 3' long terminal repeats of myc-associated proviruses appeared to be structurally intact in most tumors, although the 5' long terminal repeats were not involved in expression of either U5-myc transcripts or detectable steady-state viral RNAs. A complex array of repeated sequence elements surrounded the junctions of the internal deletions in two myc-associated proviruses. The organization of the deleted proviruses was similar to that of deleted unintegrated viral molecules, consistent with a model in which deletions occurred prior to integration.

Avian Leukosis Virus↗

PubMed1996

Evaluation of nucleic acid amplification methods for the detection of hog cholera virus.

A blind panel was tested in a diagnostic evaluation of a reverse transcription (RT) polymerase chain reaction (PCR) method for detecting hog cholera virus (HCV) from pig tissues. The capability of the RT-PCR test to discriminate between HCV and related pestiviruses, bovine viral diarrhea virus (BVDV), and those viruses causing similar diseases in swine, including African swine fever virus (ASFV) and pseudorabies virus (PRV), was also considered. Nucleic acid extraction involved either kit-based or conventional phenol:chloroform:isoamyl alcohol methods. A single-round PCR assay, using primers that hybridize to the conserved p120 nonstructural gene region, was 82.5% sensitive (n = 17) and 100% specific (n = 18) in the detection of the presence of HCV RNA. However, the sensitivity was increased to 100% following a second PCR test. In all, 4 HCV, 7 BVDV, 2 ASFV, and 1 PRV isolates were studied. Novel nucleic acid sequences were generated for 9 HCV strains. Analysis of a portion of the p120 region using these methods was suitable for HCV isolate characterization.

African Swine Fever Virus↗

PubMed1997

Biochemical and immunohistochemical characterization of human type XIX defines a novel class of basement membrane zone collagens.

Nineteen types, the product of 33 genes, comprise the collagen family of proteins. Types I, II, III, V, and XI constitute the fibrillar collagens, whereas types IV, VI to X, and XII to XIX represent the structurally diverse, nonfibrillar members. Type XIX collagen was discovered from the sequence of rhabdomyosarcoma cDNA clones. The type XIX chain consists of 1142 amino acids that contribute primarily to a unique five subdomain triple-helical region. To characterize the protein, to determine the tissue distribution, and to provide some insight into its function, we generated two type XIX-specific polyclonal antibodies. One was directed against a recombinant molecule containing amino-terminal sequences, and the second was derived from a synthetic peptide corresponding to most of the short carboxy terminus. These antibodies were used in immunoblot assays of rhabdomyosarcoma cell/matrix homogenates to identify a 165-kd disulfide-bonded and bacterial collagenase-sensitive protein. Immunohistochemical analysis of type XIX collagen was performed for human skeletal muscle, spleen, prostate, kidney, liver, placenta, colon, and skin. In contrast to Northern blot hybridizations, which showed very low levels of the 12-kb transcript in few tissues, the protein was found in all tissues examined. The type XIX collagen distribution was restricted to vascular, neuronal, mesenchymal, and some epithelial basement membrane zones, which is similar to the profile recently established (Ref. 8) and further extended here for type XV collagen. Nevertheless, localization of type XIX exhibited significant differences from type XV collagen that were particularly evident in the kidney, liver, and spleen. This report, in conjunction with the type XV results and other studies of type XVIII collagen, indicates the existence of a new collagen subgroup founded on their widespread presence in basement membrane zones regardless of chain homology. In addition to their role in basement membrane-stromal interactions, the pronounced vascular association suggests involvement of these related collagen types with angiogenic and pathological processes.

Basement Membrane↗

PubMed1989

G-proteins and second messengers in mitogenesis.

Growth factors stimulate mitogenesis via the generation of signal molecules or events. Transduction of these messages into the cell proceeds by the binding of the mitogen to a specific cell surface receptor which then stimulates the effector system (enzyme, ion channel, etc.) via a guanine nucleotide binding regulatory protein (G-protein). The specificity of a particular G-protein is a function of its unique alpha-subunit. This article reviews the effects of different growth factors upon second messenger generation and discusses the involvement of the different G-proteins in these signal transduction pathways.

Amino Acid Sequence↗

PubMed1990

Generation of transforming growth factor-alpha from the cell surface by an O-glycosylation-independent multistep process.

The precursor for transforming growth factor-alpha (TGF-alpha) is a membrane glycoprotein that can establish contact with epidermal growth factor/TGF-alpha receptors on adjacent cells or can be cleaved to release TGF-alpha that diffuses into the medium. Cleavage of pro-TGF-alpha occurs at Ala/Leu-Ala/Leu-Ala-Val-Val sites located at each end of the mature TGF-alpha sequence. To characterize the cleavage process of pro-TGF-alpha and the role of glycosylation in this process, we have introduced a pro-TGF-alpha expression vector in wild type Chinese hamster ovary (CHO) cells and in the mutant CHO cell clone ldlD that has a reversible defect in protein glycosylation. Analysis of metabolically labeled and cell surface-labeled products immunoprecipitated with antibodies directed against the extracellular TGF-alpha sequence and the cytoplasmic pro-TGF-alpha C-terminal domain shows that cleavage of pro-TGF-alpha in wild type CHO cells occurs in two steps. Both processing steps occur after pro-TGF-alpha reaches the cell surface. In the first step, pro-TGF-alpha rapidly (t1/2 = 30 min) loses the amino-terminal segment that precedes the TGF-alpha sequence. In the second step, pro-TGF-alpha is cleaved at the carboxyl terminus of the TGF-alpha sequence releasing this factor into the medium. This second step is slow (t1/2 = 2 h). The action of pancreatic elastase added to CHO-TGF-alpha cells mimics the first step but not the second one. Synthesis, cell surface exposure, rate of cleavage, and generation of bioactive TGF-alpha in ldlD-TGF-alpha cells are not markedly affected by the lack of N-acetylgalactosamine-dependent protein O-glycosylation or galactose-dependent glycan chain modification. The results indicate that, despite their similarity in amino acid sequence, the two cleavage sites that flank TGF-alpha may be processed with different kinetics which can lead to retention of pro-TGF-alpha on the cell surface.

Amino Acid Sequence↗

PubMed2004

Combined use of cultivation-dependent and cultivation-independent methods indicates that members of most haloarchaeal groups in an Australian crystallizer pond are cultivable.

Haloarchaea are the dominant microbial flora in hypersaline waters with near-saturating salt levels. The haloarchaeal diversity of an Australian saltern crystallizer pond was examined by use of a library of PCR-amplified 16S rRNA genes and by cultivation. High viable counts (10(6) CFU/ml) were obtained on solid media. Long incubation times (> or =8 weeks) appeared to be more important than the medium composition for maximizing viable counts and diversity. Of 66 isolates examined, all belonged to the family Halobacteriaceae, including members related to species of the genera Haloferax, Halorubrum, and Natronomonas. In addition, isolates belonging to a novel group (the ADL group), previously detected only as 16S rRNA genes in an Antarctic hypersaline lake (Deep Lake), were cultivated for the first time. The 16S rRNA gene library identified the following five main groups: Halorubrum groups 1 and 2 (49%), the SHOW (square haloarchaea of Walsby) group (33%), the ADL group (16%), and the Natronomonas group (2%). There were two significant differences between the organisms detected in cultivation and 16S rRNA sequence results. Firstly, Haloferax spp. were frequently isolated on plates (15% of all isolates) but were not detected in the 16S rRNA sequences. Control experiments indicated that a bias against Haloferax sequences in the generation of the 16S rRNA gene library was unlikely, suggesting that Haloferax spp. readily form colonies, even though they were not a dominant group. Secondly, while the 16S rRNA gene library identified the SHOW group as a major component of the microbial community, no isolates of this group were obtained. This inability to culture members of the SHOW group remains an outstanding problem in studying the ecology of hypersaline environments.

Archaea↗