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S A Krawetz

Publications and source records attributed to S A Krawetz.

90 records · Page 5Linked to original sources
PubMed1989

Primer-mediated enzymatic amplification of cytomegalovirus (CMV) DNA. Application to the early diagnosis of CMV infection in marrow transplant recipients.

A nucleic acid amplification procedure, the polymerase chain reaction (PCR), has been used to establish a diagnostic assay for the identification of cytomegalovirus (CMV) immediate-early sequences in clinical specimens. Preliminary testing against virus-infected cell cultures indicated that the PCR assay was highly CMV-specific, recognizing both wild-type and laboratory strains of CMV. There was no cross-reactivity with human DNA or with DNA from other herpes viruses. The sensitivity of the assay, using cloned CMV AD169 Eco RI fragment-J as template, was 1 viral genome per 40,000 cells. In a prospective study of CMV infection in bone marrow transplant recipients, the PCR assay correctly identified four patients with confirmed CMV infection. In three of these patients who were followed longitudinally, correlation of DNA reactivity with CMV culture and CMV antibody status over time indicated that DNA was the most sensitive marker for the diagnosis of CMV infection.

Adolescent↗
PubMed1988

A new family of repetitive, retroposon-like sequences in the genome of the rainbow trout.

We have identified a new family of interspersed, moderately repetitive DNA elements, termed the RSg-1 family, in the genome of the rainbow trout. Two of the elements examined here are situated upstream of sequences which code for trout nuclear proteins; a protamine gene (p101) and the clustered histone H4 gene. Sequence comparison of various RSg-1 elements indicated a high degree of nucleotide sequence homology between different members of the family. These repetitive elements exhibit well defined 3' ends which contain poly(A) segments preceded by the consensus polyadenylation signal AATAAA. Sequences flanking the 3' end of the poly(A) tract also conform to a consensus sequence. A similar sequence is also found flanking the 5' terminus of the element in the protamine clone p101, and thus may represent a target-site duplication generated upon insertion of the element into the genome. These characteristics, together with the heterogeneous nature of the 5' ends of the elements, are reminiscent of processed pseudogenes and retroposons such as the mammalian L1 family of interspersed repetitive elements.

Animals↗
PubMed1988

Bovine protamine genes contain a single intron. The structures of the two alleles.

In a recent paper, we described the isolation of the first bovine protamine cDNA (BPK59), demonstrating that the gene was autosomal and single copy (Krawetz, S. A., Connor, W., and Dixon, G. H. (1987) DNA 6, 45-57). We have since utilized the BPK59 clone as a probe, to isolate both alleles of the protamine gene from a bovine genomic library constructed in Charon 28. The sequenced alleles are highly homologous and code for identical mRNAs. Unlike the trout protamine genes, the bovine gene is not contiguous as it possesses a single intron. This intron divides the highly variable mammalian carboxyl domain from the conserved protamine basic core. Three tandemly repeated CG-like ("enhancer") sequences upstream from the CAAT box have been identified, separated by a conserved spacer region. Their possible role in the transcription of this P1 gene is discussed. The bovine genome immediately surrounding (approximately equal to 20 kilobase pairs) the protamine gene has been mapped and reveals that the sequence flanking the 5' segment of the gene is unique, while the sequence flanking the 3' segment is repetitive.

Alleles↗
PubMed1988

Sequence similarities of the protamine genes: implications for regulation and evolution.

With the recent availability of the primary structural data for the trout, bovine, and mouse protamine genes, a detailed comparison of their structures has been made. This has revealed extensive conservation of potentially biologically significant regions. An inverse correlation is apparent between gene copy number and the number of sequence-distinct protamines synthesized with the number of CP-box-like (CCYPCCC) putative transcription modulating sequences situated 5' to these genes. A common nucleotide sequence 5' to the CP-box-like putative transcription modulating sequence(s) at the end of a common region has been identified. It is postulated that this is the testis-specific protamine P1 transcription regulator sequence. Evidence based on sequence similarity is also provided for the existence of a primordial protamine gene and a scheme for the evolution of vertebrate protamine genes is proposed.

Amino Acid Sequence↗
PubMed1988

Computer video acquisition and analysis system for biological data.

The BIAS (Biological Image Analysis System) was developed to: (i) permit accurate entry and image processing of biological data; (ii) minimize the need for specialized hardware; and (iii) aid in the human genome mapping and other projects. The first mouse/cursor key-driven module was designed to be user interactive and readily accessible to many laboratories. It contains the DRSNDS programs which automate the entering of data in a systematic format. The types of data that can be entered utilizing this module are DNA-RNA gels from either a positive or negative Polaroid image, autoradiograms or biotinylated images from Southern, Northern and dot or slot blot hybridization analyses. The image is acquired using a video camera and then digitized for subsequent analysis. During the analysis graphical representations of the intermediate results are provided to assure user confidence. At any point within the program the user may obtain on-line help with the current task. The output displays the mol. wt of each individual component in the appropriate context. The present version of the program produces results comparable with a human interpreter for some data. Band shifting and optical density calculations are in a prototype form to permit evaluation of various techniques. Future work is directed at expanding the system's capabilities to interpret data from other biological analyses including DNA sequencing gels.

Algorithms↗
PubMed1987

Cloning of bovine P1 protamine cDNA and the evolution of vertebrate P1 protamines.

A bovine P1 protamine cDNA from a bull testis cDNA library was isolated utilizing a series of oligonucleotide probes. Sequence analysis showed that the cloned cDNA insert extended 317 bp to the poly(A) tail. The 51-residue 6750-dalton protamine primary translated protein is encoded within a 156-bp segment. The protamine sequence predicted from the cDNA sequence differs from that previously reported for the amino acid sequence of bovine protamine P1 by the insertion of the tripeptide Cys-Arg-Arg from residues 39-41 in the carboxy-terminal region of the mature protein. Consistent with previous hybridization analysis, nucleotide sequence comparisons showed that trout protamine cDNA was more closely related to that of bovine than to that of mouse. However, bovine P1 protamine cDNA shared greater sequence homology with mouse P1. A common nucleotide sequence of 30 bp is conserved among all three of these species. Primer extension analysis revealed that, as with trout protamine mRNAs, the majority of the untranslated portion of the mRNA lies 3' to the coding segment. Comparisons of their mRNA secondary structures by computer modeling indicate that the mRNAs fold back onto themselves, producing similar, extensively hydrogen-bonded, convoluted forms. These models support the view that translational regulation of protamine mRNA may be partially dependent on secondary structure. Southern analysis suggests that the bovine protamine P1 gene is not sex-linked and is present as one (or relatively few) copy within the bovine genome.

Amino Acid Sequence↗
PubMed1986

Oligonucleotides as probes for mammalian protamine mRNAs.

Protamine-like sequences have been identified in poly A(+)mRNAs from mammalian testes by the use of a common, complementary oligonucleotide (GCAGCANCKPTANCKNGCCAT; predicted from the common N-terminal amino acid sequence, MARYRCC, seen in several mammalian P1 protamines [D.J. McKay, B.S. Renaux and G.H. Dixon, Bioscience Reports 5:383-391 (1985)]). This oligonucleotide was utilized to prepare species-specific, primer-extended transcripts for use as Northern blotting probes. Analysis of the mRNA primer-extended transcripts revealed a discrete and similar set of products common to both bull and rat testis mRNAs which were distinct from those obtained from human testis mRNA. Northern analysis of total poly A(+) mRNAs from the corresponding mammalian testis was consistent with these results and suggests that bull and rat protamine mRNAs are more closely related to each other than to human protamine mRNA.

Animals↗
PubMed1986

In vitro expression of two proteins from overlapping reading frames in a eukaryotic DNA sequence.

The in vitro expression of two distinct proteins from overlapping reading frames in a sequence of rainbow trout genomic DNA has been demonstrated. In vitro transcription of DNA sequences, cloned in a plasmid under the control of Salmonella phage 6 polymerase promoter, led to the synthesis of two distinct and functional mRNAs corresponding to the protamine mRNA and also to another overlapping mRNA, termed Y. These mRNAs were translated in an mRNA-dependent rabbit reticulocyte lysate cell free system which synthesized the corresponding protein products. Similarities between the synthesized Pro-rich protein Y and three proline-rich proteins, the human salivary Pro-rich protein, the avian sarcoma virus protein P19 and the myc oncogene product, were evident and the significance of these findings is discussed. A synthetic oligonucleotide which is complementary to a sequence corresponding to a region of the Y protein mRNA, but upstream (5') of the transcribed protamine mRNA, hybridized faintly and only to trout brain RNA. However, more sensitive primer extension studies utilizing the Y-specific oligonucleotide detected several Y-related mRNAs in trout brain.

Amino Acid Sequence↗
PubMed1986

Isolation and fractionation of total nucleic acids from tissues and cells.

A simple and efficient method was devised to permit the isolation of total nucleic acids (poly(A+) and poly(A-) RNAs and DNA) from cells and tissues (e.g. testis) enriched in DNA. Chaotropic reagents (guanidine thiocyanate and LiBr) were utilized to inactivate nucleases rapidly, minimize the viscosity of the homogenization solution and to precipitate RNA selectively (LiBr). Both total and poly(A+)-enriched mRNAs were recovered in a biologically active form as demonstrated by their ability to programme an in vitro translation reaction. High molecular weight DNA (greater than 22 kilobases) was recovered from the LiBr-soluble supernatants by selective ethanol precipitation, subsequently purified and was in a form suitable for further biochemical analysis.

Animals↗
PubMed1986

Covalently linked sequencing primer linkers (splinkers) for sequence analysis of restriction fragments.

A new method for direct sequence analysis of DNA restriction fragments uses synthetic covalently linked complementary oligodeoxynucleotides, as universal sequencing primer linkers (splinkers). These splinkers were designed to contain an inverted repeat sequence which forms a double-stranded hairpin structure with a known restriction site. The splinkers were characterized by their ability to be self-ligating (dimerized) and by their restriction digest product analysis of both the monomer and dimer. They can also be ligated to dephosphorylated DNA restriction fragments which contain the appropriate end. This was evidenced by mobility shifts of the splinker-ligated restriction fragments. The splinker-ligated restriction fragments, after denaturation, form a single-stranded DNA template containing an inverted repeat sequence (from splinker) at one terminus. The splinker is thus suitably oriented and serves as a primer for dideoxy nucleotide (nt) sequencing catalyzed by either Klenow fragment of Escherichia coli DNA polymerase I or avian myeloblastosis virus reverse transcriptase. As demonstrated for both pBR322 and phi X174, release of the primer extension strand by digestion at the splinker restriction site results in a ladder of labelled fragments which corresponds to a unique nt sequence.

Base Composition↗
PubMed1986

A vector-primer-cloner-sequencer plasmid for the construction of cDNA libraries: evidence for a rat glyceraldehyde-3-phosphate dehydrogenase-like mRNA and a ferritin mRNA within testis.

We have developed a vector-primer-cloner-sequencer (VPCS), based on the pUC plasmids, which is easy to prepare. Stable cDNA libraries have been generated from human, rat, and bull testis. The inserts are of various size classes, including high-molecular-weight reverse transcripts which can be easily sequenced. The utility of this VPCS has been demonstrated by isolating a rat ferritin and glyceraldehyde-3-phosphate dehydrogenase-like clone.

Animals↗
PubMed1985

Tandem repeats of a specific alternating purine-pyrimidine DNA sequence adjacent to protamine genes in the rainbow trout that can exist in the Z form.

We have located an extensive (AC)n-rich but specific sequence downstream of three rainbow trout protamine genes. Although sharing considerable sequence homology, including a perfectly conserved 46 base pair repeat, the sequences exhibit a regular heterogeneity in the length of the (AC)n-rich tracts. Radioimmunoassay experiments, S1 nuclease sensitivity studies, two-dimensional electrophoretic analysis, and immunoelectron microscopy studies have been used to determine if the region could assume a Z DNA conformation. It was found that, in a supercoiled plasmid, the (AC)n-rich region has the ability to attain the Z DNA conformation under physiological conditions.

Animals↗
PubMed1985

Bovine elastin cDNA clones: evidence for the occurrence of a new elastin-related protein in fetal calf ligamentum nuchae.

Poly (A+) mRNA was isolated from fetal calf ligamentum nuchae and used for the construction of cDNA libraries. A fraction highly enriched in elastin mRNA was used to prepare the cDNA probes for screening the libraries. A 2 kb clone, pRE1, gave the most positive signal in colony hybridization. It hybridized to a mRNA of the same size as reported for elastin mRNAs from chick and sheep. Hybrid-arrested translation showed that translation of mRNAs for proteins other than elastin doublet was not inhibited by pRE1. Southern blot analysis showed that pRE1 has sequence homology with pVE6 and pVE10, which were tentatively identified as elastin-related cDNA clones representing two distinct mRNAs. DNA sequence data from the 5' end of pRE1 show that the translated amino acid sequence is not typical of known elastin sequences but contains some elastin-like sequences. All of this evidence strongly suggests the occurrence in fetal calf nuchal ligament of a mRNA which codes for a previously unknown elastin-related protein.

Amino Acid Sequence↗
PubMed1984

Isolation and in vitro translation of a mammalian protamine mRNA.

mRNA was isolated from sexually mature rat, rabbit, and bovine testes. Poly(A+) and (A-) RNAs were prepared and hybridized to a rainbow-trout protamine probe. The bovine (A+) fraction showed significant hybridization compared to the other species and these related sequences were also found in total bovine DNA. Bovine mRNA programmed the in vitro synthesis of a basic protein that possessed protamine-like properties. The mRNA was fractionated by agarose-gel electrophoresis and the fractions hybridized to the trout protamine probe. A significant hybridization signal was observed corresponding to a mRNA of approximately 400 nucleotides in length which coded for the protamine-like protein. The data support the view that we have isolated a mammalian (bovine) protamine mRNA.

Animals↗
PubMed1983

In vitro translation of elastin mRNA and processing of the translated products and the signal sequence of elastin a.

Chick embryo aorta mRNAs were translated in a gel-filtered reticulocyte lysate. The translated products showed two elastin-related proteins (a and b; relative mass approximately 70 000). The translated elastin a protein was separated essentially free of the b protein by centrifugation and sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis. The a protein was then electroeluted from the SDS-polyacrylamide gel and a partial sequence was determined by automated Edman degradation. The NH2-terminal presequence of the elastin a protein is (formula; see text) The assigned sequence is identical to that reported for the b protein. Translation of chick embryo aorta mRNA in the presence of dog pancreas microsomal membranes segregated the a and b proteins into membrane vesicles and cotranslationally cleaved their respective presequences. The processed a and b elastin proteins were isolated together and NH2-terminal proline positions were determined. These are (formula; see text) These proline positions 4 and 8 are identical to those for NH2-terminal sequence for tropoelastin. This suggests that the signal peptidase removes the 24-residue signal peptide and thus directly generates the tropoelastin sequence, with no NH2-terminal prosequence as the intermediate.

Amino Acid Sequence↗
PubMedDate not supplied

Effects of transforming growth factor-beta1 on extracellular matrix gene expression by human fibroblasts from a laryngeal stenotic lesion.

Transforming growth factor-beta1 appears to play important roles in normal wound healing by increasing synthesis of extracellular matrix components. However, the role of transforming growth factor-beta1 in the production of excessive scar tissue by fibroblasts from stenotic lesions of the larynx has not been evaluated. We examined the effect of transforming growth factor-beta1 on the steady-state messenger RNA levels of elastin, alpha2(l) procollagen, and lysyl oxidase (the enzyme that cross-links both of these structural proteins) in cell cultures of diploid human fibroblasts established from fetal skin, newborn foreskin, and an adult laryngeal stenotic lesion. Time-course and dose-response experiments demonstrated that treatment with 500 pmol/L transforming growth factor-beta1 for 20 hours induced maximal levels of mRNA for elastin (7- to 59-fold) and alpha2(l) procollagen (1.7- to 2.4-fold) in all three cultures of fibroblasts. Transforming growth factor-beta1 also increased levels of lysyl oxidase mRNA in fibroblasts cultured from newborn foreskin (2.4-fold) and a stenotic lesion (10-fold) but had minimal effects on the fibroblasts cultured from fetal skin (1.1-fold), which constitutively expressed high levels of lysyl oxidase mRNA. Furthermore, the fibroblast culture established from a laryngeal stenotic lesion responded with the highest fold-induction for all three mRNAs. Inhibition of mRNA synthesis by actinomycin D showed that transcription was required for transforming growth factor-beta1 induction of elastin, alpha2(l) procollagen, and lysyl oxidase mRNA in all three cultures of fibroblasts. Inhibition of protein synthesis by cycloheximide showed that translation was required for maximal induction by transforming growth factor-beta1 of elastin mRNA but had no observable effect on alpha2(l) procollagen mRNA in all three cultures of fibroblasts. In addition, translation was required for maximal induction of the lysyl oxidase mRNA by transforming growth factor-beta1 in the fibroblasts cultured from a stenotic lesion but not for fibroblast cultures established from fetal and adult skin. These results show that transforming growth factor-beta1 coordinately increases mRNA levels for the structural extracellular matrix proteins collagen and elastin, as well as for the cross-linking enzyme, lysyl oxidase. These data also support the hypothesis that transforming growth factor-beta1 may contribute to the formation of laryngeal stenotic lesions.

Journal Article↗
PubMedDate not supplied

Extended analysis of the region encompassing the PRM1-->PRM2-->TNP2 domain: genomic organization, evolution and gene identification.

The human male haploid expressed protamine 1 (PRM1)-->protamine 2 (PRM2)-->transition protein 2 (TNP2) locus comprises a coordinately regulated multigenic domain. This region of 16p13.13 has been used as a model to address how the organization of genes and genic domains within the human genome may influence tissue specific gene expression. Toward this goal, we have completed an extensive computational and biological analysis of the region encompassing the PRM1-->PRM2-->TNP2 domain. These analyses have revealed the likely genesis of this domain. Interestingly, the SOCS-1 gene and an hnRNPC-class pseudogene lies just 3' of this domain. Regions of nuclear matrix attachment also mark these newly identified genes.

Chromosomal Proteins, Non-Histone↗