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PubMed2001

A "loop entropy reduction" phage-display selection for folded amino acid sequences.

As a step toward selecting folded proteins from libraries of randomized sequences, we have designed a 'loop entropy reduction'-based phage-display method. The basic premise is that insertion of a long disordered sequence into a loop of a host protein will substantially destabilize the host because of the entropic cost of closing a loop in a disordered chain. If the inserted sequence spontaneously folds into a stable structure with the N and C termini close in space, however, this entropic cost is diminished. The host protein function can, therefore, be used to select folded inserted sequences without relying on specific properties of the inserted sequence. This principle is tested using the IgG binding domain of protein L and the lck SH2 domain as host proteins. The results indicate that the loop entropy reduction screen is capable of discriminating folded from unfolded sequences when the proper host protein and insertion point are chosen.

Amino Acid Sequence↗

PubMed1998

Characterization of transcript processing of the gene encoding precerebellin-1.

Precerebellin-1 (Cbln1) is a cerebellum-specific protein that shares significant sequence identity with the globular domains of the complement components C1qA, B and C, suggesting some common aspects of function and/or structure. As the C1q complex is composed of heterotrimers of C1qA, B and C it was hypothesized that multiple precerebellins may exist in a ternary complex. Northern blotting for cbln1 revealed multiple bands that could represent further family members or alternatively spliced variants. To discriminate these alternatives, probes derived from different regions of the cbln1 gene were used to identify and clone the transcripts detected on Northern blots. Four independent transcripts were repeatedly cloned from an adult mouse cerebellum cDNA library. Upon sequencing, all of these clones were found to be derived from the cbln1 gene and no additional precerebellin-related genes were isolated. Moreover, these clones accounted for the four cbln1-hybridizing bands (1.9, 2. 2, 3.2 and 5.5 kb) detected on Northern blots of adult cerebellum RNA. With one possible exception, these clones were all derived through alterations in the 3'-untranslated region (3'-UTR) of cbln1 that did not affect the coding sequence. This was achieved by the use of two polyadenylation sites and alternative (non-canonical) splicing in the 3'-UTR. Some additional variation in mRNA structure is provided by the use of alternative transcription start sites in cbln1. The possible significance of this level of diversity in the 3'-UTR is discussed.

3' Untranslated Regions↗

PubMed2003

Comparing gene expression profiles in human liver, gastric, and pancreatic tissues using full-length-enriched cDNA libraries.

In the post-genome-sequencing era, full-length cDNA-sequence resources are extremely useful for functional analyses of genes. In addition, comprehensive gene profiling of human tissues at the mRNA level is also useful in understanding the molecular mechanisms of tissue-specific functions and disease pathogenesis. In this study, to obtain a wide variety of full-length cDNA clones derived from digestive tissues, numerous expressed sequence tags were generated from libraries enriched with full-length cDNAs. In total, 13575 sequences were obtained from three cDNA libraries, which were constructed from tissues and cell lines of human liver, stomach, and pancreas. The integration of overlapping clones categorized the sequences into 5936 clusters (1666, 2746, and 2222 clusters in the liver, stomach, and pancreas, respectively). Of these, 1138 clones were scored as full-length cDNAs. Surprisingly, the redundant clones from all three tissues were assembled to show that only 101 genes (1.7% of the assembled 5936 genes) were shared. These results suggest that functional differences between tissues are probably related to their divergent gene expression profiles, and form a basis for understanding the molecular mechanisms underlying tissue-specific pathogenesis that are expressed in different organs. In addition, the full-length cDNAs obtained in this study should prove useful for future functional analyses of the genes expressed in digestive tissues.

Journal Article↗

PubMed1990

Insulin-receptor tyrosine kinase and glucose transport.

We identified the earliest events in autophosphorylation of the insulin receptor after insulin addition. Insulin-stimulated autophosphorylation at specific sites in the tyrosine kinase domain of the receptor's beta-subunit is correlated kinetically with activation of kinase-catalyzed phosphorylation of a model substrate (reduced and carboxyamidomethylated lysozyme; RCAM-lysozyme). To identify these sites, the deduced amino acid sequence of the 3T3-L1 adipocyte insulin receptor of the mouse was determined. Insulin-induced activation of substrate phosphorylation was shown to require autophosphorylation of three neighboring tyrosines (Tyr1148, Tyr1152, and Tyr1153) in the mouse receptor. A search for cellular substrates of the receptor kinase revealed that insulin causes accumulation of a 15,000-Mr phosphorylated (on tyrosine) cytosolic protein (pp15) in 3T3-L1 adipocytes treated with oxophenylarsine (PAO). PAO blocks turnover of the phosphoryl group of pp15, causing its accumulation, and thereby appears to interrupt signal transmission from the receptor to the glucose-transport system. Two membrane-bound protein phosphotyrosine phosphatases that are inhibited by PAO and are apparently responsible for the turnover of the pp15 phosphoryl group have been purified from 3T3-L1 adipocytes and characterized. These and other results support the hypothesis that turnover of the phosphoryl group of pp15, a product of insulin-receptor tyrosine kinase action, couples signal transmission to the glucose-transport system. [32P]pp15 was purified to homogeneity from 3T3-L1 adipocytes. Amino acid and radiochemical sequence analysis of the purified tryptic [32P]phosphopeptide revealed that pp15 is the phosphorylation product of 422(aP2) protein, a 15,000-Mr adipocyte protein whose cDNA we previously cloned and sequenced. 422(aP2) protein was found to bind fatty acids. When exposed to a free fatty acid, notably oleic acid, 422(aP2) protein becomes an excellent substrate of the isolated insulin-receptor tyrosine kinase. Compelling evidence indicates that on binding fatty acid, 422(aP2) protein undergoes a conformational change whereby Tyr19 becomes accessible to the receptor tyrosine kinase and undergoes O-phosphorylation. Adipose tissue and skeletal and heart muscle, which exhibit insulin-stimulated glucose uptake, express a specific insulin-responsive glucose transporter. A cDNA (GT2) that encodes this protein was isolated from a mouse 3T3-L1 adipocyte library and sequenced. We also isolated and characterized the corresponding mouse gene GLUT4. DNase I footprinting with nuclear extracts from 3T3-L1 cells revealed that a differentiation-specific nuclear factor binds to the GLUT4 promoter. The purified transcription factor C/EBP binds at the same position.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1995

The appearance of proopiomelanocortin early in vertebrate evolution: cloning and sequencing of POMC from a Lamprey pituitary cDNA library.

A proopiomelanocortin (POMC)-like hormone has been cloned and sequenced from a pituitary cDNA library of upstream migrant (prespawning) sea lamprey, Petromyzon marinus. The clone, designated LPP-1, consisted of 986 nucleotides, with an open reading frame of 277 amino acids, including a signal peptide of 22 amino acids. Like POMCs from more recently evolved vertebrates, lamprey POMC contained domains which corresponded to alpha-MSH, ACTH, and beta-endorphin. However, sequences corresponding to gamma- and beta-MSH are absent or likely nonfunctional, respectively, in this cDNA. Northern blot analyses showed low but detectable expression levels of LPP-1 in larvae and strong expression in parasitic adults and prespawning animals. These observations indicate that a recognizable POMC, distinct from proenkephalin, has an ancient lineage within subphylum Vertebrata, likely dating back to the last common ancestor of the lamprey and gnathostome lines.

Adrenocorticotropic Hormone↗

PubMed2004

Preparation of single rice chromosome for construction of a DNA library using a laser microbeam trap.

We report the development of a laser micromanipulation system and its application in the isolation of individual rice chromosomes directly from a metaphase cell. Microdissection and flow sorting are two major methods for the isolation of single chromosome. These methods are dependent on the techniques of chromosome spread and chromosome suspension, respectively. In the development of this system, we avoided using chromosome spread and cell suspension was used instead. The cell wall of metaphase rice cell was cut by optical scissors. The released single chromosome was captured by an optical trap and transported to an area without cell debris. The isolated single chromosome was then collected and specific library was constructed by linker adaptor PCR. The average insert size of the library was about 300 bp. Two hundred inserts of chromosome 4 library were sequenced, and 96.5% were aligned to the corresponding sequences of rice chromosome 4. These results suggest the possible application of this method for the preparation of other subcellular structures and for the cloning of single macromolecule through a laser microbeam trap.

Chromosomes, Plant↗

PubMed1990

Cloning and sequencing the gene encoding Escherichia coli ribonuclease I: exact physical mapping using the genome library.

The amino acid (aa) sequence of the N terminus of Escherichia coli RNase I was determined. A mixed oligodeoxynucleotide coding for that sequence was used to probe the 476 lambda clones of Kohara et al. [Cell 50 (1987) 495-508]. DNA from these clones carry almost the entire E. coli chromosome in overlapping segments. Two overlapping clones hybridized to the probe sequence. From one of them DNA containing the rna gene was subcloned and sequenced. The inferred protein contains 245 aa residues and has an Mr of 27,156, which agrees with earlier estimates from sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. RNase I is close to twice the size of pancreatic RNase A, but both enzymes contain eight Cys and four His; those aa are important for structure and function of RNase A. Proximal to the rna gene is a sequence that would code for a 23-aa peptide which conforms to consensus rules for signal peptides, and thus should transport this periplasmic enzyme. Sites for eight restriction enzymes had been mapped on each lambda clone. By relating to the map for that specific region, it was possible to position the rna gene exactly at 659 kb from the thr locus (time zero on a time scale of 100 min). This physical mapping gave a more precise (exact) map position based on distance than was possible using genetic mapping based on a time scale derived from conjugation, and should be applicable for mapping many other E. coli genes.

Amino Acid Sequence↗

PubMed1996

Construction and characterization of three region-specific microdissection libraries for human chromosome 18.

Three region-specific libraries for the entire human chromosome 18 were constructed using microdissection and Mbol linker-adaptor microcloning techniques. The libraries included 18pter-p11.1 (designated 18P library), 18q11.1-q12.3 (18Q1 library), and 18q21.1-qter (18Q2 library). Samples of the microclones from each library were analyzed in detail. The insert sizes ranged between 50-600 bp, with a mean of 180-220 bp for the three libraries. The libraries contained approximately 40-60% microclones with unique sequence inserts. More than 30 unique sequence microclones from each library were analyzed by Southern blot hybridization to demonstrate that they are human specific and were derived from chromosome 18. The human genomic HindIII fragments hybridized to each microclone were determined and microclones cross-hybridized to rodent species were identified. These region-specific libraries and the unique sequence microclones from the libraries are useful reagents for (1) isolating highly polymorphic microsatellite markers for refined linkage analysis, (2) identifying corresponding YAC, BAC or other clones with large inserts for contig assembly and high resolution physical mapping, (3) isolating cDNA clones from the dissected region, and (4) convenient sequencing of the microclones to prepare high density markers and sequence-tagged sites (STSs). Such applications have been demonstrated in a series of similarly constructed microdissection libraries from other regions of the human genome.

Chromosome Mapping↗

PubMed2001

Conformational diversity versus nucleic acid triplex stability, a combinatorial study.

The stability of a triple helix formed between a DNA duplex and an incoming oligonucleotide strand strongly depends on the solvent conditions and on intrinsic chemical and conformational factors. Attempts to increase triple helix stability in the past included chemical modification of the backbone, sugar ring, and bases in the third strand. However, the predictive power of such modifications is still rather poor. We therefore developed a method that allows for rapid screening of conformationally diverse third strand oligonucleotides for triplex stability in the parallel pairing motif to a given DNA double helix sequence. Combinatorial libraries of oligonucleotides of the requisite (fixed) base composition and length that vary in their sugar unit (ribose or deoxyribose) at each position were generated. After affinity chromatography against their corresponding immobilized DNA target duplex, utilizing a temperature gradient as the selection criterion, the oligonucleotides forming the most stable triple helices were selected and characterized by physicochemical methods. Thus, a series of oligonucleotides were identified that allowed us to define basic rules for triple helix stability in this conformationally diverse system. It was found that ribocytidines in the third strand increase triplex stability relative to deoxyribocytidines independently of the neighboring bases and position along the strand. However, remarkable sequence-dependent differences in stability were found for (deoxy)thymidines and uridines.

Base Sequence↗

PubMed2003

Diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes from groundwater and aquifer microorganisms.

To test our hypothesis that microbial autotrophic CO2 fixation plays an important role in subsurface systems of two large groundwater remediation projects, several anaerobic/microaerobic aquifer and groundwater samples were taken and used to investigate the distribution and phylogenetic diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) large-subunit genes. Two primer sets were designed for amplifying partial-subunit genes of RubisCO forms I and II from the DNA, directly extracted from the samples. PCR products were used to construct five clone libraries with putative RubisCO form I sequences, and two libraries of DNA amplified by form II primers. Selected clones were screened for variation by restriction fragment length polymorphism analysis, and a total of 28 clone inserts were sequenced and further analyzed. The phylogenies constructed from amino acid sequences derived from the partial RubisCO large-subunit sequences showed a distinct pattern. Diverse sequences affiliated to the cluster of green-like type IA RubisCO sequences were found, representing various obligate and facultative chemolithoautotrophic Proteobacteria, whereas type II RubisCO sequences detected were most closely related to those of thiobacilli species. An isolate obtained from aquifer enrichment culture, which has been provisionally named Halothiobacillus sp. RA13 on the basis of its 16S rDNA sequence, was found to contain both types of RubisCO genes, i.e., forms I and II. Physiological and ecological considerations are discussed in the context of additional microbial data and physicochemical properties.

Adaptation, Physiological↗

PubMed2005

Screening differentially expressed genes in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis.

AIM: To screen genes differentially expressed in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis. METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with high potential of lymphatic metastatic Hca-F and its synogenetic cell line Hca-P with a low metastatic potential was constructed by suppression subtracted hybridization(SSH) method. The screened clones of the subtracted library were sequenced and GeneBank homology search was performed. RESULTS: Fourteen differentially expressed cDNA fragments of Hca-F were obtained with two novel genes. CONCLUSION: SSH is a useful technique to detect differentially expressioned genes and an effective method to clone novel genes.

Animals↗

PubMed1990

A genomic clone encoding a cryptophyte phycoerythrin alpha-subunit. Evidence for three alpha-subunits and an N-terminal membrane transit sequence.

A genomic library of Chroomonas (Cryptophyceae) DNA has been constructed in lambda EMBL4. Using a synthetic oligomer as a hybridization probe, a clone containing a phycoerythrin alpha-subunit has been obtained and sequenced. The principal alpha 1- and alpha 2-subunits of the holoprotein have been partially sequenced by sequential Edman degradation and differ from the DNA derived sequence, providing evidence for at least 3 alpha-subunit genes. The nucleotide sequence of the alpha-subunit gene is GC rich and encodes an N-terminal extension which is putatively thylakoid-lumen directing.

Amino Acid Sequence↗

PubMed1998

Properties and function of the P type ion pumps cloned from Helicobacter pylori.

Three distinct P type pumps were cloned from H. pylori 69A. Two of these pumps, ATPase 439 and ATPase 948 (CopA), were isolated by gene library screening using DNA oligonucleotide primers. Amino acid similarities found for the predicted proteins were about 50% to Cd2+/Cu2+ pumps. Gene disruption mutagenesis rendered the H. pylori knockout mutants more sensitive to Zn2+ and Cd2+ (ATPase 439) or Cu2+ (CopA). Some of the ATPase 439-deficient mutants were negative for urease activity while the majority of the mutants remained positive. Functional diversity of the pumps was also reflected by the ion affinities found for N-terminal peptides of CopA to Cu2+ and of ATPase 439 to Ni2+, Cu2+ and CO2+. The membrane domain of the two pumps were experimentally shown to consist of eight membrane spans. When ATPase 439 was expressed under control of a tac promoter in Escherichia coli, vanadate-sensitive phosphate accumulation was observed cytochemically along the membrane of the host cells. The third P type pump (ATPase 115) which also exhibited homology to transition metal ATPase was identified by sequencing a library of H. pylori membrane genes. The hydropathy plot of this pump was very similar to the former H. pylori ATPases whereas the N-terminal ion binding region was distinct. It was concluded that, in H. pylori, the presence of three transition metal ATPases with distinct ion specificity contributes to the adaptive mechanisms for gastric survival.

Adenosine Triphosphatases↗

PubMed2004

A new potato virus in a new lineage of picorna-like viruses.

On constructing a cDNA library for potato, 'contaminating' sequences with a significant identity to Apple latent spherical virus (ALSV) were found. Determination of the remaining genome sequence indicated the presence of a bipartite virus with an RNA1 and 2 of 7034 and 3315 nucleotides, respectively, excluding a poly(A)tail. RNA1 encodes a single polyprotein (233 kDa) and shares highest amino acid identity with ALSV at 65%. Conserved amino acid motifs typical for helicase, protease and RNA-dependent polymerase (RdRp) functions are present. RNA2 encodes a single polyprotein (106 kDa) with amino acid identities to the flat apple isolate of Cherry rasp leaf virus (CRLV-FA) (97%) and ALSV (70%), suggesting this is a potato strain of CRLV (CRLV-pot). Phylogenetic analysis using the RdRp region shows that this virus falls within a group separate from the Comoviridae that includes members of the Sequiviridae and the taxonomically unassigned viruses ALSV, Strawberry mottle virus, Satsuma dwarf virus and Navel orange infectious mottling virus. Other regions of the genome have highest identities with both plant and animal infecting members of the picorna-like virus superfamily. The evolutionary context of CRLV-pot and related viruses is discussed. Similar viral sequences from an EST library of peppermint are also analysed.

5' Untranslated Regions↗

PubMed2006

Mining the oocyte transcriptome.

Mammalian folliculogenesis and oocyte physiology are complex and not fully understood. However, major advances over the past 15 years in our ability to create and study in vivo models have improved our understanding of these essential physiological processes. More recently, the availability of vast arrays of DNA sequence information in the forms of "complete" genomes, expressed sequence tag libraries and microarray data from reproductive tissues have stimulated the discovery of new information through genome scanning, prediction programs and in silico screening techniques. These technological improvements will help to expand our understanding of folliculogenesis and oocyte physiology and improve human reproductive health.

Bone Morphogenetic Protein 15↗

PubMed2005

[Screening of differentially expressed genes in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis].

OBJECTIVE: To screen genes differentially expressed in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis. METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with high potential of lymphatic metastasis Hca-F and its synogenetic cell line Hca-P with low metastatic potential was constructed by suppression subtractive hybridization (SSH) method. The screened clones of the subtracted library were sequenced and GenBank homology search was performed. RESULTS: Ten differentially expressed cDNA fragments of Hca-F with high potential of lymphatic spreading were obtained, two of which were newly identified ones. CONCLUSION: SSH is a useful technique to detect genes of differential expression and an effective method to clone novel genes.

Animals↗

PubMed1994

Expression of cone transducin, Gz alpha, and other G-protein alpha-subunit messenger ribonucleic acids in pancreatic islets.

The G-proteins are a family of heterotrimeric guanine nucleotide-binding proteins that play important roles in signal transduction and whose expression is regulated in a tissue-specific manner. Here we have surveyed the expression of G-protein alpha-subunits in mouse pancreatic islets. Degenerate oligonucleotide primers corresponding to conserved primary sequences in known G alpha-subunits were used in a reverse transcriptase-polymerase chain reaction, and the amplified complementary DNA (cDNA) fragments were subcloned and sequenced. Over 100 clones were analyzed, from which we determined that islet cells express at least seven G alpha-subunits: G8 alpha, Gi1 alpha or Gi3 alpha, Gi2 alpha, G11 alpha, G14 alpha, Gz alpha, and Gt2 alpha (cone transducin). In particular, the identification of Gz alpha and Gt2 alpha was of interest in that previous studies had indicated that the expression of Gz alpha was restricted mainly to the brain, retina, and adrenal gland, whereas Gt2 alpha was expressed predominantly in retinal cone photoreceptors. By Western blot analysis, we estimated that the amount of Gz alpha protein present in mouse islets was about 40% of that in retina. To further investigate the expression of Gt2 alpha, mouse Gt2 alpha cDNA was cloned from a retinal library and sequenced. The cDNA was used as a probe for Northern blot analysis, and the results confirmed that mouse islets contained a substantial level of Gt2 alpha messenger RNA (mRNA), albeit less than that found in retina (approximately 5-fold lower). Gt2 alpha mRNA was also shown to be present in a clonal mouse pancreatic alpha-cell line (alpha TC1-6) as well as in adrenal gland, pituitary, and a clonal mouse anterior pituitary cell line (AtT20). In situ hybridization revealed that Gt2 alpha mRNA was expressed essentially throughout the islet, suggesting that it is normally expressed in the abundant islet beta-cells and possibly others. In situ analysis also showed that Gt2 alpha mRNA expressed in the pituitary was limited to the intermediate and anterior lobes. We conclude that islet cells express multiple G-proteins, including several that are normally expressed at high levels in certain neuronal cells.

Amino Acid Sequence↗

PubMed1988

Molecular cloning and characterization of the DNA of two papillomaviruses from monkeys.

Benign and malignant lesions from monkeys were analyzed for the presence of papillomavirus (PV) DNA. By hybridization with different PV DNA probes under conditions of lowered stringency, two tumors were found to contain PV-specific DNA sequences: (1) a cutaneous papilloma from a Colobus monkey; and, (2) a lymph node metastasis of a squamous cell carcinoma of the penis from a Rhesus monkey. Analysis of the DNA of the papilloma from the Colobus monkey indicated the presence of extrachromosomal DNA whereas analysis of DNA from the Rhesus tumor suggested the presence of integrated viral DNA. The physical size (7.8 and 8.1 kb), colinear alignment to HPV-5, and cross-hybridization with other PV types under low stringency indicate that the two genomic DNA clones represent new PV types that have been tentatively designated as Rhesus papillomavirus type 1 (RhPV 1) and Colobus guereza papillomavirus type 2 (CgPV 2). A putative viral-host DNA junction fragment was also isolated from the Rhesus genomic library. Nucleotide sequences very closely related to RhPV 1 were observed by in situ hybridization in a laryngeal carcinoma from the Colobus guereza monkey. This report communicates the finding of novel papillomaviruses associated with a benign cutaneous tumor and genital and laryngeal malignancies in non-human primates which may have significance as a putative system for the study of papillomavirus-induced genital and laryngeal malignancies in humans.

Animals↗