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Biomedical subjects

M M Waye

Publications and source records attributed to M M Waye.

At least 55 records · Page 3Linked to original sources
PubMed1994

Isolation and characterization of a cDNA that codes for a LIM-containing protein which is developmentally regulated in heart.

During our human heart cDNA sequencing project, we have obtained a novel cDNA clone which is very similar in DNA and amino acid sequences to a rat/mouse cysteine-rich intestinal protein (1). Sequence analysis has shown that this human cysteine-rich heart protein (hCRHP) is a protein of 77 amino acids and possesses a LIM motif which is considered to be able to bind zinc. Northern blot analyses have shown that its mRNA level in rat heart is regulated developmentally. We have expressed hCRHP in E. coli using pAED4 as the vector and the cDNA was engineered so that the authentic protein is produced. The protein was partially purified and was shown to be a basic protein.

Adult↗
PubMed1994

Isolation of cDNA clones from an osteosarcoma-ROS17/2.8 library by differential hybridization.

We have used differential hybridization to isolate and characterize two novel cDNAs expressed in chondrocytes and some osteoblastic cells. A rat osteosarcoma ROS17/2.8 cDNA library was screened and cDNA clones hybridizing strongly to radiolabeled porcine calvaria cDNA but weakly to a control radiolabeled cDNA were isolated. Two clones were obtained--p.6.1 and p.10.15. A radiolabeled probe of p10.15 was shown to hybridize specifically to a 2.3 Kb message RNA from a chondrogenic clonal cell population from rat calvaria-RCJ 3.1C5.18, and the mRNA was downregulated by 1,25 (OH)2D3, which inhibits chondrogenesis in these cells. The other clone, p6.1, was found to hybridize to a 0.95 Kb message that is expressed in rat liver, kidney, lung, muscle, and brain, but not expressed in spleen and expressed only in low levels in thymus.

Amino Acid Sequence↗
PubMed1993

Biotin-containing protein as a cause of false positive clones in gene probing with streptavidin/biotin.

Using a streptavidin/biotin labeling technique, we unintentionally cloned a gene encoding a biotin carboxyl carrier protein, a subunit of biotin-dependent enzymes, from a genomic library of Streptococcus mutans strain UT-041. In colony lifts, the clone reacted positively to the streptavidin-containing detection system but could not be detected in Southern blot analysis. The amino acid sequence of the gene product, deduced from its nucleotide sequence, demonstrated all the features common to biotin carboxyl carrier proteins from other bacteria, indicating that the biotin carboxyl carrier protein in the clone had produced a "false-positive" (DNA probe-independent) reaction by binding to the streptovidin. To circumvent this problem with the detection system in gene probing in the future, we recommend that all positive clones be screened by direct incubation with streptavidin-alkaline phosphatase (SA-AP) in the absence of biotin-labeled probe DNA. Clones binding to SA-AP would be considered false positives.

Acetyl-CoA Carboxylase↗
PubMed1992

Changes in expression of alpha 1 type 1 collagen and osteocalcin mRNA in osteoblasts and odontoblasts at different stages of maturity as shown by in situ hybridization.

We investigated whether the expressed phenotype of osteoblasts and odontoblasts is changing with increasing maturity of the cells. Thus we determined, using in situ hybridization techniques, whether the expression of mRNA's for osteocalcin and the alpha 1 type 1 collagen chain was different in newly developed and more mature cuboidal osteoblasts of the primary and secondary spongiosa of radii of 8 day old rats, in mature cuboidal and older flat osteoblasts in the metatarsals of pig embryos, and in apical and coronal odontoblasts of the developing unerupted molars of pig embryos. The results indicate that newly differentiated osteoblasts in the primary spongiosa of the 8 day rat radius contained approximately the same amount of type 1 collagen message as more mature osteoblasts in the secondary spongiosa. Osteocalcin mRNA, on the other hand, was undetectable in the newly differentiated osteoblasts but clearly detectable in the mature osteoblasts of the secondary spongiosa. When we compared expression of osteocalcin and collagen type 1 mRNA in mature cuboidal and older flat osteoblasts, we found that the amount of osteocalcin mRNA relative to collagen type 1 mRNA was higher in flat osteoblasts than in cuboidal osteoblasts. In odontoblasts, however, the steady state level of collagen type 1 mRNA was higher in the older coronal odontoblasts, and the level of osteocalcin message lower, when compared to the younger apical odontoblasts. The results indicate that relative levels of osteocalcin and collagen mRNA in osteoblasts and odontoblasts vary depending on the stage in their secretory lifetime. This heterogeneity of the osteoblast and odontoblast population suggests that the composition of the matrix produced by these cells also differs.

Animals↗
PubMed1991

A phagemid vector library for cloning DNA with four-nucleotide 5' or 3' overhangs.

A phagemid vector library for cloning DNA with four nucleotide 5' or 3' overhangs has been constructed. This library is based on the pT7T3 vector (Pharmacia) which is a modification of the phagemid pTZ18U vector. We have chosen pT7T3 as the parent vector because it can be used for Sanger's dideoxy sequencing and for the generation of RNA probes with either the T7 or T3 promoter. Each member of the cloning vector series pBM has recognition sites for both of the restriction enzymes BspM1 and BstX1 in addition to the basic multiple cloning sites. BspM1 recognizes the sequence 5'...ACCTGC NNNN/NNNN...3' whereas BstX1 recognizes the sequence 5'...CCAN NNNN/NTGG...3'. Thus these two sites can be overlapped, so that only 256 vectors (instead of 512 vectors) need be constructed to cover all the theoretical possible combinations of sites which give complementary cohesive ends for cloning DNA with four nucleotide 5' or 3' overhangs. This vector library can be used for amplification cloning of DNA in a tandem array by choosing appropriate vectors which have nonpalindromic sequences. We have obtained approximately 200 members of the 256 possible clones and have organized the vectors using a MacIntosh HyperCard program for easy retrieval.

Base Sequence↗
PubMed1990

Isolation of a human vimentin cDNA with a long 3'-noncoding region from a human osteosarcoma cell line (MG-63).

Human vimentin cDNA clones were isolated and sequenced from a lambda gt11 library of the human osteosarcoma cell line MG-63. The 3'-noncoding region of the sequence is 325 nucleotides (nt) long. This cDNA sequence includes both the further 104 nt and the polyadenylation signal site at the 3' end which were earlier predicted from the genomic nt sequence, but had not been found in any previously cloned cDNAs.

Amino Acid Sequence↗
PubMed1990

An M13 vector library for cloning DNA with four nucleotide 3' overhangs.

A flexible M13 vector library incorporating the BstXI site has been developed. DNA cut by any currently commercially available restriction endonuclease that generates a 4-nucleotide (nt) 3' overhang can be ligated into a specific clone of the library. The BstXI enzyme recognizes a 6-bp bipartite palindromic sequence. The central nucleotides are not specified, and form a 4-base, 3' overhang when cut by BstXI. 5' CCANNNNN NTGG GGTN NNNNNACC 5' Since the 4-base overhang formed is not part of the BstXI recognition sequence, it is possible to generate a library of 256 different clones by introducing the BstXI site, 151 of the possible 256-member library have been isolated, including all 13 M13BF clones in which the overhang formed by BstXI digestion is complementary to those formed by currently available restriction endonucleases. Of these 13 vectors, BstXI digestion of six clones results in nonpalindromic cohesive ends and should facilitate in vitro tandem gene amplification. The BstXI site is adjacent to the four codons corresponding to the factor Xa recognition sequence. Hence the vector library could facilitate the expression of a fusion protein that could be proteolytically cleaved by factor Xa.

Base Sequence↗
PubMed1990

A Macintosh program for the management of biological samples generated by recombinant DNA techniques.

A series of Macintosh HyperCard Stacks have been developed to organize DNA clones. Four different levels of organization have been designed: the individual data cards including links to restriction maps and literature reference cards, the boxes in which the individual test tubes are organized, the freezers in which the boxes are kept and the laboratories where the freezers are found. A data base of all the commercially available DNA cloning vectors has been developed to illustrate how these stacks can be used to organize clones according to a functional hierarchy.

DNA Restriction Enzymes↗
PubMed1989

Primary structure and possible origin of the non-glycosylated basic proline-rich protein of human submandibular/sublingual saliva.

Human submandibular/sublingual saliva contains one non-glycosylated basic proline-rich protein whereas parotid saliva contains multiple such components. The submandibular protein has a primary structure identical with the C-terminal segment [TZ] of the human parotid acidic proline-rich proteins that contain 150 amino acid residues (Mr 16,000). Northern-blot analyses of human parotid and submandibular glands revealed that mRNAs containing the HaeIII repeat sequence typical for acidic proline-rich proteins are expressed in both of these salivary glands whereas mRNAs for non-glycosylated basic proline-rich proteins containing a typical BstN1 repeat sequence are expressed in the parotid but not in the submandibular gland. Products of translation in vitro of mRNAs from human parotid and submandibular glands were also examined. Two immunoprecipitable bands with Mr 29,000 and 28,000 were obtained by translation of both parotid and submandibular mRNA. In the presence of microsomal membranes these proteins gave rise to proteins electrophoretically identical with the secreted acidic proline-rich proteins of Mr 16,000. These proteins were cleaved by kallikrein, giving rise to proteins with electrophoretic mobilities identical with those of a smaller acidic proline-rich protein with Mr 11,000 and peptide TZ. Additional immunoprecipitable bands with Mr ranging from 35,000 to 46,000 were seen when parotid mRNA was used for translation in vitro, and are believed to be precursors of the basic proline-rich proteins encoded by the BstN1 repeat type mRNA. Neither these bands nor a separate precursor for the basic non-glycosylated proline-rich protein was detected when submandibular mRNA was used for translation in vitro. It is suggested that the non-glycosylated basic proline-rich protein present in human submandibular saliva arises by cleavage of acidic proline-rich proteins.

Amino Acid Sequence↗
PubMed1989

Loss of alpha I type I collagen gene expression in rat clonal bone cell lines is accompanied by DNA methylation.

Four clonal cell lines subcloned from a clonal population of fetal rat calvaria cells show a loss of type I collagen synthesis. Northern blot analysis showed that the level of alpha 1(I) collagen mRNA expression in each of the clonal populations parallels the level of collagen protein expression in each of these cell lines. The methylation pattern of the collagen gene in these clonal cell lines was determined using the restriction endonucleases MspI and HpaII. It was found that the loss in collagen type I expression correlated positively with the degree of methylation of alpha 1(I) procollagen genes, indicating that methylation of CpG may be an important mechanism of collagen gene regulation.

Animals↗
PubMed1988

A novel method for generating a nested set of unidirectional deletion mutants using mixed oligodeoxynucleotides.

A novel method that allows introduction of unidirectional deletions into cloned DNA is described. This method is based on the use of a mixture of oligodeoxynucleotide primers that have fixed 5' ends defining the end point of the deletion and variable 3' ends composed of mixtures of all four nucleotides at six positions. The 5' ends of the oligodeoxynucleotides are hybridized to a fixed location of the M13K11RX templates and the 3' ends are hybridized randomly to the DNA to be analyzed. Such oligodeoxynucleotide primers when extended with DNA polymerase can direct deletions of intervening parts of the single-stranded DNA that by design contains multiple EcoK sites; the deletion products are selected on a host strain with the EcoK restriction system (e.g., using JM101 cells). This method is an efficient way of generating a nested set of deletion mutants useful for dideoxy-sequencing. It can be used for creating a set of deletion mutants with a particular codon at the 5' or 3' end point.

Bacteriophages↗
PubMed1986

A transcription terminator in the 5' non-coding region of the tyrosyl tRNA synthetase gene from Bacillus stearothermophilus.

The 5' non-coding region of the tyrosyl-tRNA synthetase gene (tyrS) of Bacillus stearothermophilus is 324 nucleotides long. It contains a premature terminator and a strong promoter: these were identified in vitro by RNA run-off experiments and in Escherichia coli by construction of specific mutants. The terminator consists of a stem and loop structure followed by the string of T residues characteristic of rho-independent termination. This is preceded by another stem and loop structure which may permit the formation of an anti-terminator. Neither the promoter nor the premature terminator appears to be regulated by the tyrosyl-tRNA synthetase in vitro or in E. coli.

Amino Acyl-tRNA Synthetases↗
PubMed1985

EcoK selection vectors for shotgun cloning into M13 and deletion mutagenesis.

For shotgun cloning into M13 vectors, a double-stranded cassette of synthetic oligonucleotides containing a SmaI site within the two halves of an EcoK site, has been introduced into the vector M13mp8. Cloning of blunt end DNA into the SmaI site destroys the EcoK site, and recombinants are therefore preferentially selected on transfection into a K strain of E.coli. For deletion mutagenesis using synthetic oligonucleotides, an M13 vector with four copies of the EcoK cassette has been made to facilitate the joining of lacZ or a Factor Xa cleavage site to any protein reading frame.

Base Sequence↗