The modified bitegard: a method for administering supplemental oxygen and measuring carbon dioxide

HUGE is a database for human large proteins newly identified by Kazusa cDNA project, which aims to predict protein primary structures from sequences of human large cDNAs (>4 kb). In particular, cDNA clones capable of coding for large proteins (>50 kDa) are current targets of the project. More than 700 sequences of human cDNAs (average size, 5.1 kb) have been determined to date and deposited in the public databases. Notable information implied from the cDNAs and the predicted protein sequences can be obtained through HUGE via the World Wide Web at URL http://www.kazusa.or.jp/huge     

Cloning of cDNA and genomic structure of the mouse gamma-glutamyl transpeptidase-encoding gene

We have isolated and characterized cDNA and genomic clones containing the coding region for the mouse gamma-glutamyl transpeptidase (GGT). The sequences of the full-length cDNAs for three of the seven known mouse Ggt RNAs (types I, II and III) were determined and found to be identical in the coding region. Comparisons of the deduced amino-acid sequence of mouse GGT with that of rat and human reveal 95 and 79% overall identities, respectively. The mouse Ggt gene has 12 coding exon and spans approx. 12 kb. We have also re-analyzed rat genomic Ggt clones previously isolated by us and found that the rat and mouse genes share the same intron/exon boundaries. Our findings are of interest because they define the structure of the mouse and rat Ggt genes and will allow comparison with human GGT genes which, recent findings suggest, have diverged substantially from rodents.     

Gene sequence tags from Plasmodium falciparum genomic DNA fragments prepared by the "genease" activity of mung bean nuclease

A genes-first approach to genome sequencing is described which efficiently generates gene sequence tags from genomic DNA. Mung bean nuclease (EC 3.1.30.1) cleaves the genomic DNA of many organisms before and after genes and within some introns. Analysis of gene sequence tags prepared from mung bean nuclease-digested Plasmodium falciparum DNA demonstrates that this method has several advantages over the popular cDNA expressed sequence tag approach. To date, 673 sequence tags containing over 215 kb of sequence have been generated from 400 clones. Sixty clones (15%) have significant similarity to sequences in the protein and translated nucleic acid data bases. These represent 51 unique genes, of which only 5 encode previously known P. falciparum proteins. The identified proteins include those expressed in erythrocytic, exoerythrocytic, and gametocytic stages of the parasite. Thirty percent of clones identified appear to carry complete coding regions. The spacer DNA separating genes is rarely cloned. These gene sequence tags will form a useful data base from which to initiate projects to develop new therapeutics, vaccines, and strategies to control human malaria.     

Molecular cloning and functional expression of feline thrombopoietin

Feline thrombopoietin (TPO) was molecularly cloned to establish a basis for cytokine therapy of thrombocytopenia in cats. cDNA clones covering the whole coding sequence of feline TPO were isolated from feline liver. The feline TPO cDNA obtained in this study contained an open reading frame encoding 349 amino acid residues. The predicted amino acid sequence of feline TPO shared 78.7, 69.9, 72.9 and 83.0% similarity with sequences of human, murine, rat and canine TPO, respectively. Four cysteine residues and two of four N-glycosylation sites that are conserved among species were also found at the corresponding positions in feline TPO. The feline TPO cDNA fragment encoding the whole amino acid coding region was recloned into an expression vector, and the resulting vector was transfected into 293T cells using the calcium phosphate method. The supernatant of the transfected 293T cells stimulated the proliferation of a human megakaryoblastic leukemia cell line (UT-7/TPO) cells in a dose dependent manner, indicating that the feline TPO cDNA obtained in this study encodes biologically active feline TPO.     

Sex determination of infant and juvenile skeletons: I. Morphognostic features

The human neutrophil-derived cationic peptide HP-4 exhibits corticostatic activity on adrenal cells and is an L-type calcium channel agonist at nanomolar concentrations. Complementary DNA clones encoding the HP-4 precursor have been isolated from a human bone marrow cDNA library by screening with oligonucleotide probes. The nucleotide sequence shares about 72% identity with the cDNA encoding defensin HP-1, but differs from it, and from other genes of this family characterized to date, by an extra 83-base segment. This extra segment is not adjacent to an intron and is apparently the result of a recent duplication within the coding region corresponding to most of the mature HP-4 peptide. The predicted amino acid sequence shows the HP-4 precursor structure to be typical of this family of molecules. By analysis of DNA from a pannel of hamster/human hybrid cell lines, the HP-4 gene was found to be on chromosome 8, as is the gene for human peptide HP-1. Comparison with the few sequences of other corticostatin/defensin genes available does not indicate distinct lineages of corticostatic and noncorticostatic peptides, since HP-1 and HP-4 cDNA sequences share more identity with each other than either shares with cDNAs encoding rabbit MCP-1 or MCP-2, or guinea pig GNCP-1.     

Molecular characterization and localization of human metabotropic glutamate receptor type 4

Oligonucleotides of consensus sequences from rat metabotropic glutamate receptor (mGluR) genes were synthesized and used to amplify human DNA by the polymerase chain reaction (PCR). Five unique human sequences homologous to these rat receptor genes were isolated including mGluR4. A human cerebellum cDNA library was screened using this amplified mGluR4 sequence as a probe and yielded clones which between them contained the complete coding sequence for human mGluR4. The coding sequence is very similar to the equivalent rat gene (90% DNA sequence identity and 97% predicted protein sequence identity). The mGluR4 cDNA was transfected in Chinese hamster ovary (CHO) cells and stable clonal cell lines were isolated. Stimulation of the expressed receptor by L-2-amino-4-phosphonobutyrate (L-AP4), L-glutamate or (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) resulted in a reduction of forskolin-stimulated cyclic AMP (cAMP) with EC50 values of 0.2, 13 and 90 microM respectively. Quisqualate had little effect at concentrations up to 1 mM. In Northern blots mGluR4 mRNA appears to be brain-specific, and shows a distinct distribution (excluding the cerebellum), being expressed in the thalamus, hypothalamus and caudate nucleus. In situ hybridization studies on human brain sections confirmed this general pattern of distribution. The strongest mGluR4 mRNA signal was found in the cerebellar granule cells consistent with the reported distribution of mGluR4 in the rat brain. The major difference from the rat brain is the presence in the human brain of mGluR4 mRNA in the caudate nucleus and putamen.     

Effect of demographic factors, urinary peak flow rates, and Boyarsky symptom scores on patient treatment choice in benign prostatic hyperplasia

Thiopurine methyltransferase (TPMT) is a genetically polymorphic enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine. This genetic polymorphism is an important factor responsible for individual variation in thiopurine drug response. A cDNA for TPMT has been cloned from T84 human colon carcinoma cells. Northern blot analysis of multiple human tissues was performed with the T84 human colon carcinoma TPMT cDNA open reading frame (ORF) as a probe as one step toward understanding the molecular basis for the TPMT genetic polymorphism. Three mRNA species (approximately 1.4, 2.0, and 3.6 kb in length) were present in all tissues studied, including liver. However, none of these mRNAs matched the length of the 2.7 kb T84 TPMT cDNA. Therefore, it was important to clone a TPMT cDNA from a human drug-metabolizing organ such as the liver to determine whether its sequence matched that of the cDNA cloned from the T84 cell line. A human liver cDNA library was screened with the T84 TPMT cDNA ORF as a probe, and a 1.8 kb cDNA was isolated with a coding region sequence identical to that of the T84 TPMT cDNA. The TPMT cDNA ORF was then used to screen a human lymphocytes genomic DNA library in an attempt to clone the TPMT gene(s) in humans. Three intronless clones were isolated with identical ORF sequences that were 96% identical to that of the TPMT cDNA, but which contained multiple nucleotide substitutions and one deletion. The 3'- and 5'-flanking regions of one of the genomic DNA clones were sequenced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and expression of the cDNA coding for the human myelin/oligodendrocyte glycoprotein

We report here the characterization of a full-length cDNA encoding the human myelin/oligodendrocyte glycoprotein (MOG). The sequence of the coding region of the human MOG cDNA is highly homologous to that of other previously cloned mouse, rat, and bovine MOG cDNAs, but the 3' untranslated region differs by an insertion of an Alu sequence between nucleotides 1,590 and 1,924. Accordingly, northern blot analyzes with cDNA probes corresponding to the coding region or the 3' untranslated Alu-containing sequence revealed a single band of 2 kb, rather than the 1.6 kb of bovine, rat, or mouse MOG cDNA(s). Immunocytochemical analysis of HeLa cells transfected with human MOG cDNA, which was performed using a specific antibody raised against whole MOG, clearly indicated that MOG is expressed at the cell surface as an intrinsic protein. These data are in accordance with the predicted amino acid sequence, which contains a signal peptide and two putative transmembrane domains. The knowledge of the human MOG sequence should facilitate further investigations on its potential as a target antigen in autoimmune demyelinating diseases like multiple sclerosis.     

Primary structure of the kinase domain region of rabbit skeletal and cardiac muscle titin

A 2.3 kb region of rabbit cardiac and skeletal muscle titin has been cloned. The cDNA sequences of the two tissues are identical and show 91% identity on the nucleotide level with the corresponding region of human cardiac muscle titin. On the amino acid level the identity is 96% and similarity is 98%. Alignment of predicted amino acid sequences of several homologous kinase domains reveals that the rabbit titin kinase has all the necessary elements of an active catalytic domain and carries a potential regulatory region on its C-terminal end. The distance of the 2.3 kb contig from the 3' end of the message was determined to be 5.7 kb in both tissues using oligonucleotide directed RNase H cleavage of titin mRNAs. This is essentially identical with the length of the fully sequenced human cardiac titin C-terminal end. It therefore appears unlikely that there are major tissue specific differences in this 8 kb cDNA region which encodes the C-terminus of rabbit skeletal and cardiac titin.     

Assembly of the zygotic centrosome in the fertilized Drosophila egg

Large-scale cDNA analysis provides several great advantages for genome investigations in rice. Isolated and partially characterized cDNA clones have contributed not only to the construction of an RFLP linkage map and physical maps of the chromosomes but also to investigations of the mechanisms of expression of various isozymes and family genes. The ultimate aim of our large-scale cDNA analysis is to catalogue all the expressed genes of this important cereal, including tissue-specific, developmental stage-specific, and stress-specific genes. As of August 1996, the Rice Genome Research Program (RGP) has isolated and partially sequenced more than 29,000 cDNA clones from various tissues and calluses in rice (Nipponbare, a japonica variety). The sequence data were translated into amino acid sequences for the 3 possible reading frames, and the similarity of these amino acid sequences to known proteins registered in PIR were examined. About 25% of the clones had significant similarities to known proteins. Some of the hit clones showed library-specific distributions, indicating that the composition of the clones in each library reflects, to some extent, the regulation of gene expression specific to differentiation, growth condition, or environmental stress. To further characterize the cDNA clones, including unknown clones, nucleotide sequence similarities of 24,728 clones were analyzed and the clones were classified into around 10,000 independent groups, suggesting that around a half or one third of expressed genes in rice have already been captured. These results obtained from our large-scale cDNA analysis provide useful information related to gene expression and regulation in rice.     

Suppression in transformed avian fibroblasts of a gene (crp) encoding a cysteine-rich protein containing LIM domains

Using cDNA subtraction and differential hybridization techniques, a cDNA library derived from normal quail embryo fibroblasts was screened for clones corresponding to genes whose expression was suppressed in v-myc-transformed, as compared with normal, quail embryo fibroblasts. One of the isolated cDNA clones corresponded to a 0.9-kb mRNA that was present in normal quail and chicken embryo fibroblasts, but was virtually absent from all transformed avian cells tested: quail embryo fibroblasts transformed by the v-myc, v-myc/v-mil or v-src oncogenes, cells derived from a methylcholanthrene-induced quail fibrosarcoma or v-myc-transformed chicken macrophages. Nucleotide sequence analysis of the original and supplementary cDNA clones indicated that the corresponding gene encodes a 194 amino acid cysteine-rich protein (M(r) 20,911). A database search revealed that the gene is the avian homolog of a human primary response gene (crp) of unknown function. Both the quail and human CRP proteins contain two copies of a cysteine-rich amino acid sequence motif (LIM) with putative zinc-binding activity that was previously identified in several proteins with presumed regulatory functions essential for cell growth or differentiation.     

Mammalian mitochondrial ribosomal proteins. N-terminal amino acid sequencing, characterization, and identification of corresponding gene sequences

The integrity of healthy mitochondria is supposed to depend largely on proper mitochondrial protein biosynthesis. Mitochondrial ribosomal proteins (MRPs) are directly involved in this process. To identify mammalian mitochondrial ribosomal proteins and their corresponding genes, we purified mature rat MRPs and determined 12 different N-terminal amino acid sequences. Using this peptide information, data banks were screened for corresponding DNA sequences to identify the genes or to establish consensus cDNAs and to characterize the deduced MRP open reading frames. Eight different groups of corresponding mammalian MRPs constituted from human, mouse, and rat origin were identified. Five of them show significant sequence similarities to bacterial and/or yeast mitochondrial ribosomal proteins. However, MRPs are much less conserved in respect to the amino acid sequence among species than cytoplasmic ribosomal proteins of eukaryotes and bacteria.     

Predicting effective drug concentrations for individual patients. Determinants of pharmacodynamic variability

To study the diversity of protocadherins, a rat brain cDNA library was screened using a cDNA for the cytoplasmic domain of human protocadherin Pcdh2 as a probe. The resultant clones contained three different types. One type corresponds to rat Pcdh2; the other two types are distinct from Pcdh2 but contain the same sequence in their cytoplasmic domains and part of the 3' flanking sequence. To clarify the structure of the proteins defined by the new clones, a putative entire coding sequence corresponding to one of the clones was determined. The overall structure is essentially the same as Pcdh2, indicating that the proteins defined by this clone, and probably by other clones, belong to the protocadherin family. Two PCR experiments and an RNase protection assay showed the existence of the corresponding mRNAs in rat brain preparations. Human and mouse cDNA clones with the same sequence properties were also isolated. Taken together, these results indicate that the clones are not cloning artifacts and that corresponding mRNAs are actually expressed in brains of various species. The results of in situ hybridization showed that the mRNAs corresponding to these clones were expressed in different regions in brain. Since protocadherins encoded by these mRNAs are likely to have different specificity in their interaction and share a common activity at their cytoplasmic domains, these protocadherins may provide a molecular basis, in part, to support the complex cell cell interaction in brain.