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Primary structure and functional properties of an epithelial K channel.

Expression cloning in Xenopus oocytes was used to identify a clone for a renal K channel. The clone, named ROMK2, was obtained from a cDNA library constructed in the plasmid vector pSPORT using size-selected poly(A)+ RNA from whole rat kidney. ROMK2 consists of 1,837 nucleotides, with an open reading frame of 1,116 bases predicted to code for a 372-amino acid peptide. The clone appears to be a splice variant of a recently reported K channel (ROMK1) from rat renal outer medulla (Ho, K.H., C.G. Nichols, W.J. Lederer, J. Lytton, P.M. Vassilev, M.V. Kanazirska, and S.C. Hebert. Nature Lond. 362: 31-37, 1993). Northern blot analysis indicates that ROMK2 is expressed in renal cortex, medulla, and papilla. Expression in other tissues appears to be much lower. The functional properties of the channel as measured in Xenopus oocytes indicate its close relationship to ROMK1 and more distant relationship to the inward rectifier K channel (IRK1) (Kubo, Y, T.J. Baldwin, Y. N. Jan, and L. Y. Jan. Nature Lond. 362: 127-133, 1993). The inward conductance of the channel is a saturable function of external K, with a half-maximal conductance at <5 mM. The selectivity sequence for ion permeability based on reversal potential measurements was K > Rb > NH4 > Na, Li. The conductance to Rb was only one-half that to K. Extracellular Ba2+ and Cs+ blocked the channel in a voltage-dependent manner. The high sensitivity of Cs+ block to voltage is consistent with the channel's operating as a multi-ion pore. The channel was blocked by high concentrations (100 microM) of glibenclamide. It did not appear to be blocked by extracellular Na+ or tetraethyl-ammonium ion. Patch-clamp measurements indicated a single-channel conductance of 30 pS in the presence of 110 mM K and high open probability that was weakly dependent on voltage. This channel may be involved in maintaining the membrane potential of renal cells and/or mediating renal K secretion.

Amino Acid Sequence

Verbal recoding of visual stimuli impairs mental image transformations.

Two experiments were carried out to test the hypothesis that verbal recoding of visual stimuli in short-term memory influences long-term memory encoding and impairs subsequent mental image operations. Easy and difficult-to-name stimuli were used. When rotated 90 degrees counterclockwise, each stimulus revealed a new pattern consisting of two capital letters joined together. In both experiments, subjects first learned a short series of stimuli and were then asked to rotate mental images of the stimuli in order to detect the hidden letters. In Experiment 1, articulatory suppression was used to prevent subjects from subvocal rehearsal when learning the stimuli, whereas in Experiment 2, verbal labels were presented with each stimulus during learning to encourage a reliance on the verbal code. As predicted, performance in the imagery task was significantly improved by suppression when the stimuli were easy to name (Experiment 1) but was severely disrupted by labeling when the stimuli were difficult to name (Experiment 2). We concluded that verbal recoding of stimuli in short-term memory during learning disrupts the ability to generate veridical mental images from long-term memory.

Attention

Isolation, expression, and mutation of a rabbit skeletal muscle cDNA clone for troponin I. The role of the NH2 terminus of fast skeletal muscle troponin I in its biological activity.

A cDNA for rabbit fast skeletal muscle troponin I (TnI) was isolated and sequenced. The clone contains a coding sequence predicting a 182-amino-acid protein with a molecular mass of 21,162 daltons. The translated sequence is different from that reported by Wilkinson and Grand (Wilkinson, J. M., and Grand, R. J. A. (1978) Nature 271, 31-35) in that Arg-153, Asp-154, and Leu-155 must be inserted into their original sequence. Amino acid sequencing of adult rabbit TnI confirmed this result. In order to investigate the role of the NH2 terminus of TnI in its biological activity, we have expressed a recombinant deletion mutant (TnId57), which lacks residues 1-57, in a bacterial expression system. Both wild type TnI (WTnI) and TnId57 inhibited acto-S1-ATPase activity and this inhibition could be fully reversed by troponin C (TnC) in the presence of Ca2+. Additionally both WTnI and TnId57 bound to an actin affinity column. Thus, both inhibitory actin binding and Ca(2+)-dependent neutralization by TnC were retained in TnId57. TnC affinity chromatography was used to compare the binding of TnI and TnId57 to TnC. Using this method, two types of interaction between TnC and TnI were observed: 1) one which is metal independent (or structural) and 2) one dependent on Ca2+ or Mg2+ binding to the Ca(2+)-Mg2+ sites of TnC. The same experiments with TnId57 demonstrated that the type 1 interaction was weakened, and type 2 binding was lost. This method also revealed an interaction between TnC and TnI which is dependent upon Ca2+ binding to the Ca(2+)-specific sites of TnC and which is retained in TnId57. Taken together, these results suggest that the NH2 terminus of TnI may constitute a Ca(2+)-Mg(2+)-dependent interaction site between TnC and TnI and play, in part, a structural role in maintaining the stability of the troponin complex while the COOH terminus of TnI contains a Ca(2+)-specific site-dependent interaction site for TnC as well as the previously demonstrated Ca(2+)-sensitive inhibitory and actin binding activities.

Actins

Bacillus subtilis alkaline phosphatases III and IV. Cloning, sequencing, and comparisons of deduced amino acid sequence with Escherichia coli alkaline phosphatase three-dimensional structure.

Bacillus subtilis has an alkaline phosphatase multigene family. Two members of this gene family, phoAIII and phoAIV, were cloned, taking advantage of in vitro constructed strains containing a plasmid insertion within one or the other of the structural genes. The DNA sequences of the two genes showed approximately 64% identity at the DNA level and 63% identity in the deduced primary amino acid sequences. The phoAIII and phoAIV genes code for predicted proteins of 47,149 and 45,935 Da, respectively. Comparison of the deduced primary amino acid sequence of the mature proteins with other sequenced alkaline phosphatases from Escherichia coli, yeast, and humans shows 25-30% identity. Based on the refined crystal structure of E. coli alkaline phosphatase, it appears that the active site and the core of the structure are retained in both Bacillus alkaline phosphatases. However, both proteins are truncated at the amino terminus compared with other mature alkaline phosphatases, three sizable surface loops of E. coli are deleted, and a minidomain is replaced with a larger domain in the model. Neither Bacillus alkaline phosphatase sequenced contains any cysteine residues, an amino acid implicated in intrachain disulfide bond formation in other alkaline phosphatases.

Alkaline Phosphatase

Single amino acid substitution defines a naturally occurring genetic variant of human thymidylate synthase.

Previously, we identified an altered structural form of thymidylate synthase (TS) in a human colonic tumor cell line. This form, which is encoded by a variant structural gene, renders cells relatively resistant to 5-fluoro-2'-deoxyuridine as a result of the reduced affinity of the enzyme for the active metabolite 5-fluoro-2'-deoxyuridylic acid. We have isolated a cDNA clone specific to the altered TS and have determined its sequence. Two point mutations distinguish the normal from the altered TS mRNAs. One, a (A----G) change, is located within the 3'-untranslated region; the other, a T----C change within the amino acid-coding region, predicts replacement of tyrosine by histidine at residue 33 of the polypeptide. This sequence change was confirmed by direct analysis of cDNA amplified by the polymerase chain reaction and was further verified using allele-specific oligonucleotides as probes in Northern blots. These results, along with studies by other laboratories showing Tyr33 to be evolutionarily conserved, suggest that this residue plays an important role in TS function.

Amino Acid Sequence

Bovine alpha 1----3-galactosyltransferase: isolation and characterization of a cDNA clone. Identification of homologous sequences in human genomic DNA.

We have isolated, by immunological screening of a lambda gt11 expression library, a cDNA clone that represents the complete coding sequence for bovine alpha 1----3-galactosyltransferase. The coding sequence predicts a membrane-bound protein with three distinct structural features: a large, potentially glycosylated COOH-terminal domain (346 amino acids), a single transmembrane domain (16 amino acids), and a short NH2-terminal domain (6 amino acids). Thus, the domain structure for this transferase is similar to that deduced for beta 1----4-galactosyltransferase (Shaper, N. L., Hollis, G. F., Douglas, J. G., Kirsch, I. R., and Shaper, J. H. (1988) J. Biol. Chem. 263, 10420-10428) and alpha 2----6-sialyltransferase (Weinstein, J., Lee, E. V., McEntee, K., Lai, P.-H., and Paulson, J. C. (1987) J. Biol. Chem. 262, 17735-17743). S1 analysis demonstrates that two sets of mRNAs, which are heterogeneous at their 5' ends, are transcribed. Because both sets initiate upstream of the translational start site, only one protein is encoded by this gene. alpha 1----3-Galactosyltransferase is widely expressed in different mammalian species, with the notable exception of man and Old World monkeys (Galili, U., Shohet, S. B., Kobrin, E., Stults, C.L.M., and Macher, B. A. (1988) J. Biol. Chem. 263, 17755-17762). By Northern blot analysis we were indeed unable to detect transcripts for this enzyme in various human and Old World monkey cell lines; transcripts were readily detected in other mammalian species. However, by Southern blot analysis, homologous sequences for alpha 1----3-galactosyltransferase were identified in human genomic DNA. This suggests that the gene, although present in the human genome, is normally not expressed. These observations have potential medical implications. Because many humans have high levels of circulating antibodies directed against the enzymatic product of alpha 1----3-galactosyltransferase (Gal alpha 1----3Gal beta 1----4GlcN Ac) (Galili, U., Clark, M. R., Shohet, S. B., Buehler, J., and Macher, B. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1369-1373), it has been suggested that activation of this normally silent gene may play a role in autoimmune disease in man (Etienne-Decerf, J., Malaise, M., Mahieu, P., and Winand, R. (1987) Acta Endocrinol. 115, 67-74).

Amino Acid Sequence

cDNA and gene sequence of Manduca sexta arylphorin, an aromatic amino acid-rich larval serum protein. Homology to arthropod hemocyanins.

The serum (storage) proteins produced by insect larvae at the end of the feeding cycle are hexameric blood proteins with one or more type of subunits. The cDNA and gene structure of the aromatic amino acid-rich larval serum protein arylphorin from the tobacco hornworm, Manduca sexta, has been determined. In M. sexta arylphorin there are two subunits alpha and beta, which have 686 and 687 amino acids, respectively, and whose amino acid sequences are 68% identical. The two genes, separated by 7.1 kilobases of chromosomal DNA, are transcribed in the same direction. Based on the alignment of the amino acid sequence, the rate of nucleotide substitution between the two coding regions predicts that the two genes diverged about 100 million years ago. Both genes contain 5 exons and the upstream region contains a sequence, TGATAAA, which is similar to a sequence found in all other storage protein genes for which information is available. When the National Biomedical Research Foundation protein sequence data base was searched, it was found that the arylphorin subunits showed significant similarity to the arthropod hemocyanins, which are hexameric oxygen-carrying proteins. Based on the alignment of the sequence of M. sexta arylphorin and the hemocyanin from the spiny lobster (Panulirus interruptus), for which a 3.2 A structure has been determined, it was observed that the highest concentration of conserved residues were found in those regions of the sequence which are involved in subunit interactions in the hexameric protein. It is suggested that the insect storage proteins and the arthropod hemocyanins have evolved from a common ancestor.

Amino Acid Sequence

Hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), and their receptors in human breast cells and tissues: alternative receptors.

We sought to determine whether the hepatocyte growth factor/scatter factor (HGF/SF)- and keratinocyte growth factor-receptor systems were expressed in normal breast cells, breast carcinoma cell lines, normal breast tissues, and breast cancer tissues. Reverse transcriptase-polymerase chain reaction and hot blotting were used to detect HGF, HGF/SF (met) receptor, KGF, and KGF receptor mRNAs in human mammary epithelial (HME) and stromal (HMS) cells. We also examined breast carcinoma (MDA-MB-157, SCC 38, and SCC 70) and spontaneously immortalized breast epithelial (HMT 3522) cell lines, as well as normal breast and breast carcinoma tissues. PCR products were also confirmed by nucleic acid sequencing. The effects of HGF and KGF, compared to EGF and heparin-binding EGF, on the proliferation of normal human mammary epithelial cells in serum-free defined medium was determined by cell counting. HGF and KGF mRNAs were detected in HMS cells, but not HME cells. KGF receptor mRNA was detected in HME cells, but not HMS cells. HGF/SF receptor mRNA was detected in both HME and HMS cells. mRNAs were also detected in normal breast and breast carcinoma tissues, as well as breast carcinoma and transformed breast epithelial cell lines. Alternative cDNA sequences that are predicted to code for a soluble KGF receptor and a membrane bound, truncated HGF/SF receptor were detected in breast epithelial cells and breast tissues. HGF and KGF maintained viability and stimulated proliferation of HME cells.

Adult

Cloning of the NCX2 isoform of the plasma membrane Na(+)-Ca2+ exchanger.

The Na(+)-Ca2+ exchanger is an important regulator of cellular Ca2+ levels, and one isoform of this transporter, NCX1, has been cloned previously (Nicoll, D.A., Longoni, S., and Philipson, K.D. (1990) Science 250, 562-565). We now report the cloning of a second isoform (NCX2) of the Na(+)-Ca2+ exchanger which was present in a rat brain cDNA library. NCX2 is predicted to code for a protein of 921 amino acids. NCX1 and NCX2 are 61 and 65% identical at the nucleotide and amino acid levels, respectively, and are the products of different genes. The genes for NCX1 and NCX2 are located on human chromosomes 2 and 14, respectively. Hydropathy profiles of the two exchangers are very similar. Transcripts of NCX2 are detected in brain and skeletal muscle. NCX2 was expressed in Xenopus oocytes and Na(+)-Ca2+ exchange activity was analyzed electrophysiologically by the giant inside-out, excised patch technique. Outward currents were evoked by the application of Na+ with the exchanger operating in the reversed mode (extracellular Ca2+ exchanging for intracellular Na+). The affinity for Na+ (30 mM) and the current-voltage relationship of NCX2 are similar to those for NCX1. Like NCX1, NCX2 is secondarily regulated by intracellular Ca2+, but the affinity of NCX2 for regulatory Ca2+ (1.5 microM) upon initial application of Na+ is lower than that of NCX1 (0.3 microM). The existence of multiple Na(+)-Ca2+ exchanger isoforms may provide flexibility for regulation and expression.

Amino Acid Sequence

The incidence of herpes zoster.

BACKGROUND: There are few population-based studies of the natural history and epidemiology of herpes zoster. Although a relatively common cause of morbidity, especially among the elderly, contemporary estimates of herpes zoster incidence are lacking. Herein we describe a population-based investigation of incident and recurrent herpes zoster from 1990 through 1992 in a health maintenance organization. METHODS: The health maintenance organization's automated medical records contain clinical and administrative information about care rendered to patients in ambulatory settings, emergency departments, and hospitals. Cases of herpes zoster were ascertained by screening the medical record for coded diagnoses. The predictive value of a herpes zoster diagnosis code was determined by review of a sample of patient records. Records from all patients with potential recurrences were also reviewed. RESULTS: The overall incidence, based on 1075 cases in 500,408 person-years, was 215 per 100,000 person-years (95% confidence interval, 192 to 240 per 100,000) and did not vary by gender. Although the rate increased sharply with age, approximately 5% of the cases occurred among children younger than 15 years. Infection with human immunodeficiency virus was documented in 5% of the persons with incident herpes zoster and cancer in 6%. Four persons had confirmed recurrences of herpes zoster (744 per 100,000 person-years; 95% confidence interval, 203 to 1907); three of these persons were infected with the human immunodeficiency virus. CONCLUSIONS: The recorded incidence of herpes zoster was 64% higher than that reported 30 years ago; the age-standardized rate was more than twofold higher. Immunosuppressive conditions had little impact on overall incidence, although they were strongly associated with early recurrences.

Adolescent

Extensive sequence homology of the goldfish ras gene to mammalian ras genes.

We cloned ras-related sequences from goldfish genomic libraries constructed as recombinants using the lambda phage. Restriction enzyme mapping of the clones obtained revealed three kinds of ras-related sequences among approximately 350,000 genomic clones. One of these clones was partially sequenced. Comparison with the nucleotide sequences of mammalian ras genes showed that the determined sequences covered the predicted amino acid coding regions and parts of the intervening regions. The predicted amino acid sequences of the cloned ras-related goldfish gene suggested that the coding region is localized separately in DNA, and that its exon-intron boundaries are exactly the same as those of corresponding mammalian genes. The nucleotide and amino acid sequences of the goldfish ras-related gene may have extensive homologies to mammalian p 21 protein. Among the three mammalian ras proteins, the predicted amino acid sequence of the sequenced ras-related goldfish clone is most closely homologous (96%) to the Kirsten ras protein. Differences in the predicted amino acid sequence were greatest in the sequence predicted from the fourth exon; fewer differences were found in the sequence from the third exon, and only slight or no differences were found in the sequence predicted for the first and second exons. The 12th and 61st amino acids from the N-terminal of the protein, which are thought to be critical positions for GTP binding and catalysis, are both conserved in the goldfish protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence

Cloning and sequencing the HinfI restriction and modification genes.

The HinfI restriction and modification genes were cloned on a 3.9-kb PstI fragment inserted into the PstI site of plasmid pBR322. Both genes are confined to an internal 2.3-kb BclI-AvaI subfragment. This subfragment was sequenced. Two large open reading frames (ORF's) are present. ORF1 codes for the methylase [predicted 359 amino acids (aa)] and ORF2 codes for the endonuclease (predicted 262 or 272 aa).

Amino Acid Sequence

Identification of human gene structure using linear discriminant functions and dynamic programming.

Development of advanced technique to identify gene structure is one of the main challenges of the Human Genome Project. Discriminant analysis was applied to the construction of recognition functions for various components of gene structure. Linear discriminant functions for splice sites, 5'-coding, internal exon, and 3'-coding region recognition have been developed. A gene structure prediction system FGENE has been developed based on the exon recognition functions. We compute a graph of mutual compatibility of different exons and present a gene structure models as paths of this directed acyclic graph. For an optimal model selection we apply a variant of dynamic programming algorithm to search for the path in the graph with the maximal value of the corresponding discriminant functions. Prediction by FGENE for 185 complete human gene sequences has 81% exact exon recognition accuracy and 91% accuracy at the level of individual exon nucleotides with the correlation coefficient (C) equals 0.90. Testing FGENE on 35 genes not used in the development of discriminant functions shows 71% accuracy of exact exon prediction and 89% at the nucleotide level (C = 0.86). FGENE compares very favorably with the other programs currently used to predict protein-coding regions. Analysis of uncharacterized human sequences based on our methods for splice site (HSPL, RNASPL), internal exons (HEXON), all type of exons (FEXH) and human (FGENEH) and bacterial (CDSB) gene structure prediction and recognition of human and bacterial sequences (HBR) (to test a library for E. coli contamination) is available through the University of Houston, Weizmann Institute of Science network server and a WWW page of the Human Genome Center at Baylor College of Medicine.

Algorithms

GraphyloVar: predicting the impact of non-coding variants using a multi-species sequence model.

MOTIVATION: Understanding the functional impact of genetic variants is a key problem for precision medicine. Tools like CADD, PhyloP, and PhastCons are useful, but they often look at each position in the genome in isolation. This means they can miss important information from the evolutionary history that connects different species. In this paper, we extend our previous model, Graphylo, to predict the effects of variants. Our new model, GraphyloVar, is built to directly utilize the phylogenetic tree that relates the species. RESULTS: GraphyloVar is a deep learning model that considers both DNA sequence and evolutionary patterns from many species. It uses two main components: Graph Convolutional Networks (GCNs) to process the phylogenetic tree, and Transformer encoders to extract features from the DNA sequences. Pre-trained to predict population-level allele frequencies on the TOPMed whole-genome sequencing cohort, GraphyloVar achieves an AUROC of 0.6246 zero-shot on &#x223c;149M held-out variants, and an ensemble with CADD reaches 0.6442 (+0.020, P<10-15). Fine-tuned GraphyloVar achieves the highest AUROC across all 13 MPRA benchmark datasets. By integrating deep learning with explicit phylogenetic input, GraphyloVar offers a powerful and complementary approach to variant effect prediction that utilizes the full evolutionary history from many species to better identify and prioritize important non-coding variants. AVAILABILITY AND IMPLEMENTATION: Code and datasets are available at https://github.com/DongjoonLim/GraphyloVar under DOI: 10.5281/zenodo.20616818.

Phylogeny

Chromosome-level genome assembly of Nothapodytes nimmoniana.

Nothapodytes nimmoniana is a plant species belonging to the genus Nothapodytes in the family Icacinaceae. This species holds significant medicinal value due to its camptothecin content. In this study, we present the first chromosome-level genome assembly of N. nimmoniana constructed using NGS, Hi-C, and HiFi sequencing technologies. The assembled genome spans 3.53&#x2009;Gb across 14 chromosomes, with an N50 length of 248.74&#x2009;Mb. Genome annotation revealed that repetitive sequences constitute 80.82% of the genome size, and 83,269 protein-coding genes were predicted. Additionally, 4,360,538&#x2009;bp of non-coding RNA were annotated. This genomic resource provides a foundation for further investigation into camptothecin biosynthesis pathways and plant phylogeny in N. nimmoniana.

Genome, Plant

Concordance of experimentally mapped or predicted Z-DNA sites with positions of selected alternating purine-pyrimidine tracts.

The recent electronmicroscopic and biochemical mapping of Z-DNA sites in phi X174, SV40, pBR322 and PM2 DNAs has been used to determine two sets of criteria for identification of potential Z-DNA sequences in natural DNA genomes. The prediction of potential Z-DNA tracts and corresponding statistical analysis of their occurrence have been made on a sample of 14 DNA genomes. Alternating purine and pyrimidine tracts longer than 5 base pairs in length and their clusters (quasi alternating fragments) in the 14 genomes studied are under-represented compared to the expectation from corresponding random sequences. The fragments [d(G X C)]n and [d(C X G)]n (n greater than or equal to 3) in general do not occur in circular DNA genomes and are under-represented in the linear DNAs of phages lambda and T7, whereas in linear genomes of adenoviruses they are strongly over-represented. With minor exceptions, potential Z-DNA sites are also under-represented compared to random sequences. In the 14 genomes studied, predicted Z-DNA tracts occur in non-coding as well as in protein coding regions. The predicted Z-DNA sites in phi X174, SV40, pBR322 and PM2 correspond well with those mapped experimentally. A complete listing together with a compact graphical representation of alternating purine-pyrimidine fragments and their Z-forming potential are presented.

Animals

Isolation and characterization of a partial cDNA for a human sialyltransferase.

A probe generated from the coding sequence of the rat hepatic beta-galactoside alpha 2,6-sialyltransferase was used to screen a human cDNA library constructed of human submaxillary gland mRNA lambda gt-11. We report the isolation and characterization of a human cDNA, HSM-ST1, that is putatively the human homolog of the beta-galactoside alpha 2,6-sialyltransferase. The largest human clone contains a 1.3 kb cDNA insert and is predicted to encompass 75% of the coding sequence as well as a small portion of the 3' untranslated region. Comparative analysis of this insert with the rat hepatic alpha 2,6-sialyltransferase sequence indicates 79% nucleotide similarity between the two sequences in the predicted coding region. On the amino acid level, the degree of conservation is 86%. Substantial sequence similarity is observed in the 3'-untranslated region between the rat and human sequences as well. S1 nuclease analysis was performed to demonstrate the expression of HSM-ST1 transcripts in the human hepatoma cell line, HepG2, and in the human colonic adenocarcinoma cell lines, LS174T.

Amino Acid Sequence

Chromosomal level genome assembly of medicinal plant Chrysosplenium macrophyllum.

Chrysosplenium macrophyllum Oliv., a perennial herb native to China, is widely used in traditional medicine for its notable therapeutic properties. However, the absence of a reference genome has constrained its full potential for research and application. This study presents the first chromosome-level de novo genome assembly of C. macrophyllum, constructed by integrating long reads from Oxford Nanopore Technologies (ONT), short reads from BGI, and Hi-C data. The final assembly spans 2.55&#x2009;Gb, with a scaffold N50 of 93.38&#x2009;Mb, and 83.70% of the genome has been assigned to 22 chromosomes. The mapping rate of the BGI short reads to the genome is approximately 97.94%, and BUSCO analysis reveals that 97.94% of the predicted genes are complete. A total of 62,921 protein-coding genes were predicted, with functional annotations for 93.67% of them. This chromosome-level genome assembly represents an important resource for expanding our understanding of Chrysosplenium species and supports future genomic studies and applications.

Genome, Plant