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PubMed2000

Sequence analysis and initial characterization of two isozymes of hydroxylaminobenzene mutase from Pseudomonas pseudoalcaligenes JS45.

Pseudomonas pseudoalcaligenes JS45 grows on nitrobenzene by a partially reductive pathway in which the intermediate hydroxylaminobenzene is enzymatically rearranged to 2-aminophenol by hydroxylaminobenzene mutase (HAB mutase). The properties of the enzyme, the reaction mechanism, and the evolutionary origin of the gene(s) encoding the enzyme are unknown. In this study, two open reading frames (habA and habB), each encoding an HAB mutase enzyme, were cloned from a P. pseudoalcaligenes JS45 genomic library and sequenced. The open reading frames encoding HabA and HabB are separated by 2.5 kb and are divergently transcribed. The deduced amino acid sequences of HabA and HabB are 44% identical. The HAB mutase specific activities in crude extracts of Escherichia coli clones synthesizing either HabA or HabB were similar to the specific activities of extracts of strain JS45 grown on nitrobenzene. HAB mutase activity in E. coli extracts containing HabB withstood heating at 85 degrees C for 10 min, but extracts containing HabA were inactivated when they were heated at temperatures above 60 degrees C. HAB mutase activity in extracts of P. pseudoalcaligenes JS45 grown on nitrobenzene exhibited intermediate temperature stability. Although both the habA gene and the habB gene conferred HAB mutase activity when they were separately cloned and expressed in E. coli, reverse transcriptase PCR analysis indicated that only habA is transcribed in P. pseudoalcaligenes JS45. A mutant strain derived from strain JS45 in which the habA gene was disrupted was unable to grow on nitrobenzene, which provided physiological evidence that HabA is involved in the degradation of nitrobenzene. A strain in which habB was disrupted grew on nitrobenzene. Gene Rv3078 of Mycobacterium tuberculosis H37Rv encodes a protein whose deduced amino acid sequence is 52% identical to the HabB amino acid sequence. E. coli containing M. tuberculosis gene Rv3078 cloned into pUC18 exhibited low levels of HAB mutase activity. Sequences that exhibit similarity to transposable element sequences are present between habA and habB, as well as downstream of habB, which suggests that horizontal gene transfer resulted in acquisition of one or both of the hab genes.

Amino Acid Sequence↗

PubMed1989

Molecular cloning and sequencing of a cDNA encoding N alpha-acetyltransferase from Saccharomyces cerevisiae.

Acetylation is the most frequently occurring chemical modification of the alpha-NH2 group of eukaryotic proteins and is catalyzed by an N alpha-acetyltransferase. Recently, a eukaryotic N alpha-acetyltransferase was purified to homogeneity from Saccharomyces cerevisiae, and its substrate specificity was partially characterized (Lee, F.-J. S., Lin L.-W., and Smith, J. A. (1988) J. Biol. Chem. 263, 14948-14955). This article describes the cloning from a yeast lambda gt11 cDNA library and sequencing of a full length cDNA encoding yeast N alpha-acetyltransferase. DNA blot hybridizations of genomic and chromosomal DNA reveal that the gene (so-called AAA1, amino-terminal, alpha-amino, acetyltransferase) is present as a single copy located on chromosome IV. The use of this cDNA will allow the molecular details of the role of N alpha-acetylation in the sorting and degradation of eukaryotic proteins to be determined.

Acetyltransferases↗

PubMed1998

Characterization of a gene that is inversely correlated with estrogen receptor expression (ICERE-1) in breast carcinomas.

Differential display was used to compare patterns of gene expression in two estrogen receptor (ER)-positive breast carcinoma cell lines (MCF7 and T-47D) and two ER-negative breast carcinoma cell lines (MDA-MB-231 and HBL-100). A 377-bp fragment was identified that was overexpressed in the ER-negative cell lines. Sequence analysis of this clone and comparison with the GenBank/EMBL databases indicated that it did not match any genes published previously. The expression pattern of this gene was inversely correlated with the expression of ER and has been termed ICERE-1 (inversely correlated with estrogen receptor expression). A longer clone of ICERE-1 was isolated from a MDA-MB-231 cDNA library and sequence analysis indicated that this 2168-bp cDNA contained an ORF encoding a protein of 234 amino acids that bears little similarity with any previously described protein sequence. Northern blot analysis of a panel of breast cancer cell lines demonstrated that an ICERE-1 mRNA of approximately 2.2 kb was abundantly expressed in the ER-negative breast carcinoma cell lines, MDA-MB-231 and HBL-100, and the ER-negative cell lines. HEC-1-B, HeLa, and 293. Expression of ICERE-1 was absent or minimal in the ER-positive breast carcinoma cell lines MCF7, T-47D, MDA-MB-361, ZR-75-1, BT-474 and BT-20. Reverse transcription/PCR was used to examine ICERE-1 expression in 29 primary breast carcinomas, 15 of which had been designated as ER positive and 14 as ER negative by immunohistochemistry. The expression level of ICERE-1 was significantly lower (P < 0.001) in the ER-positive tumors compared with the ER-negative tumors. The pattern of expression of ICERE-1 indicates that this gene may be involved in tumor biology specific to hormonally unresponsive breast cancers.

Amino Acid Sequence↗

PubMed1983

The L1Md long interspersed repeat family in the mouse: almost all examples are truncated at one end.

We have characterized a large repetitive element which has been found at seven different locations within the beta globin locus of the BALB/c mouse. This repeat has an unusual structure in that each of the different members has the same end of the element conserved while the other end terminates at a different point in each repeat member. The sequences within the repeats from the beta globin locus have homology with other repetitive families such as the MIF-1, Bam-5, R, and the BamH1 families. These were recently proposed (T. Fanning, (1983) Nucleic Acids Res. 11, 5073-5091) to be part of a structure with the same organization which we found in the globin locus. Probing plaques from a BALB/c genomic library with sequences derived from the repeats in the globin locus shows that virtually all of the repeats from this family are organized in a manner consistent with the proposed structure.

Animals↗

PubMed2001

Identification of the gamma-aminobutyric acid receptor beta(2) and beta(3) subunits in rat, rabbit, and human kidneys.

The properties and functions of gamma-aminobutyric acid (GABA(A)) receptors in the mammalian central nervous system are well studied. However, the presence and significance of GABA(A) receptors in nonneural tissue is less clear. The goal of this study was to examine the expression and localization of the GABA(A) receptor beta(2) and beta(3) subunits in the kidney. Reverse transcriptase products from RNA isolated from rat and rabbit kidney cortex and cerebellum and rabbit S(2) segments were amplified by use of PCR and GABA(A) beta(2) and beta(3) subunit-specific primers. Sequencing of the kidney PCR products revealed that the rat kidney cortex and rat neuronal GABA(A) receptor beta(2) subunit were identical in nucleotide composition. The rabbit kidney and rabbit neuronal GABA(A) receptor beta(2) subunit were 99% identical in nucleotide composition. Sequencing of the kidney PCR products revealed that the rat kidney cortex and rat neuronal GABA(A) receptor beta(3) subunits were 93% and 95% identical in nucleotide and amino acid composition, and rabbit kidney cortex and rabbit neuronal GABA(A) receptor beta(3) subunits were 95% and 98% identical in nucleotide and amino acid composition, respectively. PCR screening of a human kidney cDNA library and sequencing revealed that the human kidney cortex and neuronal beta(3) subunits were identical in nucleotide composition. Immunoblot analysis of rat kidney cortex and brain identified immunoreactive proteins in the 55 to 57 kD region, corresponding to the GABA(A) receptor beta(2) and beta(3) subunits. Immunohistochemistry revealed cytosolic and basolateral staining of the proximal convoluted and straight tubule. These results provide compelling evidence for the expression of the GABA(A) receptor beta(2) and beta(3) subunits in the kidney of multiple species and the localization of the beta(2)/beta(3) subunits to the renal proximal tubule.

Amino Acid Sequence↗

PubMed1997

Cloning and sequence analysis of rat fertilin alpha and beta--developmental expression, processing and immunolocalization.

Fertilin alpha and beta are members of the MDC (metalloproteinase-like, disintegrin-like, cysteine-rich) protein family and are expressed on the sperm surface where they have been proposed to play a role in mammalian fertilization. Inhibition of sperm-oocyte binding and sperm-oocyte fusion make fertilin an attractive target for the development of an immunocontraceptive vaccine. Full-length cDNAs encoding alpha and beta fertilin subunits were isolated from a rat testis cDNA library and sequenced. Using reverse transcription-polymerase chain reaction (RT-PCR), the developmental expression of fertilin alpha and beta was determined in pre-pubertal and mature rat testes. Fertilin alpha mRNA was present at all stages of development, suggesting that it is not exclusively expressed in post-meiotic germ cells. In contrast, fertilin beta mRNA was first identified in day 19 testes, coincident with the presence of pachytene spermatocytes. Polyclonal antisera raised against a 28-residue peptide (corresponding to part of the disintegrin domain) and two recombinant fusion proteins identified a 90 kDa protein in testicular sperm extracts and a 60 kDa protein in caput and cauda epididymidal sperm extracts, the predicted sizes for rat fertilin beta precursor and mature protein respectively. Indirect immunofluorescence using the anti-peptide antisera stained the acrosomal cap of permeabilized testicular, caput and caudal spermatozoa and elongating spermatids in testicular sections.

ADAM Proteins↗

PubMed1988

Gene conversion and differential regulation in the rat P-450 IIA gene subfamily. Purification, catalytic activity, cDNA and deduced amino acid sequence, and regulation of an adult male-specific hepatic testosterone 15 alpha-hydroxylase.

Previous studies on regulation of the rat hepatic P-450 IIA1 cDNA have provided evidence for a second gene closely related to but regulated in a manner quite distinct from P-450 IIA1. Experiments were carried out to isolate the cDNA for this second P-450 gene, designated IIA2, in order to study more directly its regulation and relationship to IIA1. A full length cDNA to IIA2 was isolated from an adult male rat liver lambda gt11 library and sequenced completely. The IIA2 cDNA shared 93% nucleotide and 88% deduced amino acid similarities with the previously characterized IIA1 cDNA (Nagata, K., Matsunaga, T., Gillette, J., Gelboin, H. V., and Gonzalez, F. J. (1987) J. Biol. Chem. 262, 2787-2793). The protein, deduced from the cDNA, contained 492 amino acids and a calculated Mr of 56,352. Comparison of the IIA1 and IIA2 cDNAs revealed areas of low nucleotide similarity interspersed with areas of absolute identity, suggesting that gene conversions have played a role in the evolution of the IIA subfamily. Expression of IIA1 and IIA2 mRNAs in rat liver during development was studied with use of specific oligonucleotide probes. IIA1 mRNA was increased within 1 week after birth in both male and female rats; however, its postpubertal expression was decreased in males yet remained elevated in females. In contrast, IIA2 mRNA was markedly induced in male rat liver at puberty but was not detectable in females at any age examined. Furthermore, only IIA1 mRNA was induced by treatment of rats with 3-methylcholanthrene. Although IIA1 and IIA2 mRNAs were actively expressed in hepatic tissue, no evidence for their expression was found in lung, kidney, or intestine, suggesting that the IIA genes have tissue-specific promoters. Reconstituted enzyme assays on the purified protein products P-450 IIA1 and P-450 IIA2 showed that, although both enzymes share considerable sequence similarity, their positional specificities toward the prototype substrate testosterone are strikingly different.

Amino Acid Sequence↗

PubMed2003

Formation of pseudo-terminal restriction fragments, a PCR-related bias affecting terminal restriction fragment length polymorphism analysis of microbial community structure.

Terminal restriction fragment length polymorphism (T-RFLP) analysis of PCR-amplified genes is a widely used fingerprinting technique in molecular microbial ecology. In this study, we show that besides expected terminal restriction fragments (T-RFs), additional secondary T-RFs occur in T-RFLP analysis of amplicons from cloned 16S rRNA genes at high frequency. A total of 50% of 109 bacterial and 78% of 68 archaeal clones from the guts of cetoniid beetle larvae, using MspI and AluI as restriction enzymes, respectively, were affected by the presence of these additional T-RFs. These peaks were called "pseudo-T-RFs" since they can be detected as terminal fluorescently labeled fragments in T-RFLP analysis but do not represent the primary terminal restriction site as indicated by sequence data analysis. Pseudo-T-RFs were also identified in T-RFLP profiles of pure culture and environmental DNA extracts. Digestion of amplicons with the single-strand-specific mung bean nuclease prior to T-RFLP analysis completely eliminated pseudo-T-RFs. This clearly indicates that single-stranded amplicons are the reason for the formation of pseudo-T-RFs, most probably because single-stranded restriction sites cannot be cleaved by restriction enzymes. The strong dependence of pseudo-T-RF formation on the number of cycles used in PCR indicates that (partly) single-stranded amplicons can be formed during amplification of 16S rRNA genes. In a model, we explain how transiently formed secondary structures of single-stranded amplicons may render single-stranded amplicons accessible to restriction enzymes. The occurrence of pseudo-T-RFs has consequences for the interpretation of T-RFLP profiles from environmental samples, since pseudo-T-RFs may lead to an overestimation of microbial diversity. Therefore, it is advisable to establish 16S rRNA gene sequence clone libraries in parallel with T-RFLP analysis from the same sample and to check clones for their in vitro digestion T-RF pattern to facilitate the detection of pseudo-T-RFs.

Animals↗

PubMed1990

The 75-kilodalton protein of Chlamydia trachomatis: a member of the heat shock protein 70 family?

The gene encoding a 75-kilodalton (kDa) protein of Chlamydia trachomatis was cloned, expressed, and sequenced. Genomic libraries from C. trachomatis serovar D DNA were constructed in vectors pUC18 and lambda gt11 and were screened with a panel of monoclonal antibodies against C. trachomatis antigens. The only recombinants identified were those that reacted with antibody UM-13, which has specificity for a genus-specific epitope on the 75-kDa protein. The gene was localized to a 2.9-kilobase DNA fragment and sequenced. The gene consists of a long open reading frame of 1,956 nucleotides, which translates into 652 amino acids totalling 70,558 daltons in mass. Putative promoter elements and a ribosome binding site were identified within 5'-flanking sequences, and a typical rho-independent terminator was identified within 3'-flanking sequences. Screening of the GenBank nucleic acid sequence data bank revealed extensive similarity between the chlamydial 75-kDa gene and the heat shock protein 70 (hsp70) family or proteins. In particular, 71 and 69% amino acid sequence similarities were identified with hsp70 of Escherichia coli and Bacillus megaterium, respectively. Polyclonal antibodies were produced to the recombinant antigen in rabbits and detected epitopes on elementary bodies in enzyme-linked immunosorbent and indirect microimmunofluorescence assays. Antibodies reacted with an antigen of identical molecular mass in L2 and C serovars in an immunoblot assay and neutralized these serovars in cell culture. The 75-kDa protein appears to be a chlamydial homolog of hsp70, is immunoaccessible on native elementary bodies, and is a target for neutralization.

Amino Acid Sequence↗

PubMed2000

Bone morphogenetic proteins-2 and -4: negative growth regulators in adult retinal pigmented epithelium.

PURPOSE: To determine the relative level and localization of bone morphogenetic protein (BMP-4 mRNA in the retina and retinal pigmented epithelium (RPE) under normal and pathologic conditions, to seek clues regarding possible functions. METHODS: Clones isolated from an RPE cDNA library were sequenced and used as probes for northern blot analysis. Expression in the retina and RPE was investigated in mouse models using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. The effect of recombinant proteins on RPE proliferation was investigated by thymidine incorporation. RESULTS: Bovine clones with high homology to BMP-2 and BMP4 were isolated from a subtracted RPE cDNA library. Northern blot analysis using the clones as probes demonstrated abundant and differential expression in adult bovine RPE, but with RT-PCR and in situ hybridization, expression was also demonstrated in mouse retinal neurons. In mice with oxygen-induced ischemic retinopathy there was a striking decrease in BMP-4 mRNA in the retina within 6 hours of the onset of hypoxia that was maintained for at least 5 days. In mice with inherited photoreceptor degeneration, there was a dramatic decrease in BMP4 mRNA in retina and RPE during and after the degeneration. mRNA for the type II BMP receptor was observed in freshly isolated and cultured RPE cells, isolated retina, and freshly isolated bovine aortic endothelial cells. Thymidine incorporation in early-passage RPE cells showed a 14-fold stimulation above control with 5% serum that was decreased to 322%, 393%, and 313% in the presence of BMP-2 (10 ng/ml), BMP4 (10 ng/ml), and transforming growth factor (TGF)-,1 (2 ng/ml), respectively. CONCLUSIONS: BMP-2 and BMP-4 may serve as negative growth regulators in the retina and RPE that are downregulated by injury, to allow tissue repair. Modulation of expression of the BMPs may provide a means to control the exaggerated wound repair that occurs in proliferative retinopathies.

Animals↗

PubMed2003

Construction and characterization of a vestibular-specific cDNA library using T7-based RNA amplification.

Using a very small amount of inner-ear tissue, we constructed a human vestibular cDNA library by means of T7-based amplification of RNA. This library should allow us to identify genes likely to be involved in auditory and vestibular functions. Here we first report the characterization of the human vestibular cDNA library. Among 506 cDNA clones randomly selected from the vestibular cDNA library, DNA sequences of 301 cDNA clones were identical to those of genes of known function. Twenty-two cDNA clones were considered to be novel because they did not match any cDNA sequences in the public database. The information in our study will provide a valuable resource for identifying several novel genes underlying deafness disorders and vestibular dysfunction.

Bacteriophage T7↗

PubMed2003

Identification of a member of a new RNase a family specifically secreted by epididymal caput epithelium.

In this study, we purified the first member of a new ribonuclease (RNase) A family from fluid of the proximal caput of the boar epididymis. This protein, named "Train A," is the most abundant compound secreted in the anterior part of the boar epididymis. After 2D electrophoresis, it is characterized by more than 10 isoforms ranging in size from 26 to 33 kDa and pI from 5 to 8.5. Several tryptic peptides were N-terminal sequenced, and an antiserum against one of these peptides was obtained. The protein was immunolocalized in the epididymal epithelium of the proximal caput, especially in the Golgi zone and the apical cytoplasm of the principal cells. In the lumen, spermatozoa were negative but droplets of reaction product were observed within the lumen. Full lengths of Train A cDNA were obtained from a lambdagt11 boar caput epididymis library and sequenced. The deduced protein is composed of 213 amino acids, including a 23-amino acid peptide signal and a potential N-glycosylation site. The mRNA of this protein has been retrieved and partially sequenced in the bull, horse, and ram, and homologous cDNA is found in databanks for the rat, mouse, and human. All the sequences are highly conserved between species. This protein and its mRNA are male-specific and exclusively expressed in the proximal caput of the epididymis, the only site where they have been found. Train A presents an RNase A family motif in its sequence. The RNase A family is a group of several short proteins (20-14 kDa) with greater and lesser degrees of ribonucleolytic activity and with supposed different roles in vivo. However, the presence of a long-conserved N-terminal specific sequence and the absence of RNase catalytic site for Train A indicate that Train A protein is a member of a new family of RNase A.

Amino Acid Sequence↗

PubMed2006

EST analysis of cDNA libraries from the entomopathogenic fungus Beauveria (Cordyceps) bassiana. II. Fungal cells sporulating on chitin and producing oosporein.

In the accompanying paper [Cho, E.-M., Liu, L., Farmerie, W. & Keyhani, N. O. (2006). Microbiology 152, 2843-2854], the analysis of expressed sequence tag (EST) libraries derived from homogeneous single-cell populations of aerial conidia, in vitro blastospores and submerged conidia of the entomopathogenic fungus Beauveria (Cordyceps) bassiana has been reported. Here an extended EST analysis is presented of complex cell mixtures derived from fungal cells sporulating on chitin or grown under culture conditions inducing the production of the B. bassiana secondary metabolite, oosporein. Fungal material used for the construction of the libraries included germinating conidia and blastospores, mycelia, as well as cells in various developmental stages. Approximately 2,500 5' end sequences were determined from random sequencing of clones from each library, and were clustered into 277 contigs with 1,069 singlets, and 306 contigs with 1,064 singlets, for the chitin and oosporein libraries, respectively. Almost half (45-50 %) of the sequences in each library displayed either no significant similarity (e value >10(-4)) or similarity to hypothetical proteins found in the NCBI database. Approximately 20-25 % of the sequences in each library could be annotated by gene ontology terms. A comparative analysis between the two libraries, as well as the libraries in the accompanying paper, is presented. A set of 4,360 clustered and unique sequences was characterized. The data are indicative of a highly plastic gene expression repertoire being available to B. bassiana for growth during different environmental and developmental conditions, and provides a dataset for gene discovery and genome annotation.

Benzoquinones↗

PubMed1994

Characterization of 65 bovine microsatellites.

Microsatellites or simple sequence repeat (SSR) polymorphisms are used widely in the construction of linkage maps in many species. High levels of polymorphism coupled with the ease of analysis of the polymerase chain reaction (PCR) have resulted in this type of maker being one of the most widely used for genetic analysis. In this paper we describe 58 polymorphic bovine microsatellites that were isolated from insert size selected bovine genomic libraries. Primer sequences, number of alleles, and heterozygosity levels in cattle reference families are reported. Chromosomal locations for 47 of these microsatellites as well as for 7 previously described systems derived from entries in the Genbank or EMBL databases have been determined. The markers map to 24 syntenic or chromosomal locations. Polymorphic bovine microsatellites were estimated to occur, on average, every 320 kb, and there is no evidence of clustering in the genome. Thirty of the bovine-derived microsatellite systems gave specific and polymorphic products in sheep, adding to the number of useful markers in that species.

Animals↗

PubMed1995

Identification of 3'-terminal exons from yeast artificial chromosomes.

We report an extension of 3'-terminal exon trapping technology to the identification of transcribed sequences from yeast artificial chromosomes (YACs). A 350-kb YAC containing mouse genomic DNA was gel-purified and used as the target DNA for the 3'-terminal exon trapping strategy. A novel direct ligation/transfection approach was employed to increase the efficiency of trapping 3'-terminal exons from recombinant vector-derived chimeric mRNA. The resulting RT-PCR product was then used to generate a plasmid library. Randomly chosen individual subclones from this library were sequenced, and the results indicate that 86% met sequence criteria characteristic of 3'-terminal exons, whereas 14% were background from identified sources. PCR mapping efforts suggest eight putative last exons present within this YAC, whereas RT-PCR studies demonstrate that three reside within valid expressed sequences.

Animals↗

PubMed1993

cDNA cloning, structural features, and eucaryotic expression of human TAG-1/axonin-1.

Axonal surface glycoproteins, composed of repeated immunoglobulin-like and fibronectin-type-III(FNIII)-like domains, mediate adhesion between axons or between axons and non-neuronal cells or extracellular matrix proteins. Several representatives of this group promote neurite outgrowth, when presented as substratum to neurons in culture, and have been implicated in axonal guidance mechanisms. TAG-1 and axonin-1 are presumptive species homologues of the rat and the chick, respectively; together with F11/F3, they form a subgroup of Ig/FNIII-like molecules containing a glycosyl-PtdIns membrane anchor. Recent reports on tumor suppressor genes encoding Ig-like and FNIII-like sequences prompted us to isolate the human homologue to TAG-1 and axonin-1. Polymerase chain reaction (PCR) primers were designed to regions conserved in both TAG-1 and axonin-1 using deoxyinosine at ambiguous positions. An expected 1000-bp fragment was obtained from cDNA derived from adult human cerebellum. Using this PCR fragment as a probe, several clones were isolated from a human fetal brain cDNA library. Nucleotide sequence analysis of a full-length clone, as expected, revealed a high degree of similarity to rat TAG-1 (91% identity) and chicken axonin-1 (75% identity) at the amino acid level. The encoded protein was then transiently expressed in monkey COS1 cells, and a stable mouse myeloma cell line was established expressing human TAG-1/axonin-1. The transfected COS1 and myeloma cells showed immunoreactivity on the cell surface with polyclonal anti-(chicken axonin-1) serum. On Western blots, the same antibodies recognized the recombinant protein migrating slightly slower on SDS/PAGE than chicken axonin-1. A comparison of chicken and human brain-tissue proteins by Western-blot analysis revealed a similar apparent molecular mass difference between the two species, which might be due to three additional N-glycosylation sites present on human TAG-1/axonin-1. Immunostaining of cryostat sections of embryonic retinas with polyclonal anti-(axonin-1) serum showed similar expression patterns in chicken and human samples at corresponding developmental stages. An additional shared feature of human TAG-1/axonin-1, rat TAG-1 and chick axonin-1 is their attachment to the cell membrane with a glycosyl-PtdIns anchor.

Amino Acid Sequence↗

PubMed1984

Evidence for a coding pattern on the non-coding strand of the E. coli genome.

Analysis of codon usage frequency for the combined coding sequences of 52 E. coli genes, taken from the European Molecular Biology Laboratory Nucleotide Sequence Data Library, Release 2, shows that there is a significant positive correlation between the frequency with which a given codon appears on the coding strand and the frequency with which it appears, in phase, on the non-coding strand.

Amino Acid Sequence↗

PubMed2003

Anaerobic digestion of manure and mixture of manure with lipids: biogas reactor performance and microbial community analysis.

Anaerobic digestion of cattle manure and a mixture of cattle manure with glycerol trioleate (GTO) was studied in lab-scale, continuously stirred tank reactors (CSTR) operated at 37 degrees C. The reactor codigesting manure and lipids exhibited a significantly higher specific methane yield and a higher removal of VS than the reactor treating manure. Microbial population analysis done by cultivation--most probable number (MPN) test and specific methanogenic activity (SMA) measurement, revealed higher MPN and increased SMA of methanogenic populations of biomass from the reactor codigesting manure and lipids. Spatial microbial distribution and activity was studied in digested materials fractionated into size of particles > 200 microm, 50-200 microm and 0.45-50 microm. With manure, the main pool of methanogenic activity from propionate, butyrate and hydrogen was associated with the particles > 200 microm, while the activity of acetotrophic methanogens was uniformly distributed in all fractions. When digesting manure and lipids, an enhanced methanogenesis was detected both for particles > 200 microm and the 50-200 microm fraction. The molecular methods--temperature gradient gel electrophoresis (TGGE), cloning library and sequencing of 16S rDNA--showed presence of a restricted number of archaeal species in both reactors. The vast majority of clones was phylogenetically most closely related to Methanosarcina siciliae.

Agriculture↗