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S A Krawetz

Publications and source records attributed to S A Krawetz.

At least 73 records · Page 4Linked to original sources
PubMed1993

Lysyl oxidase copper-talon complex: a model.

The translated primary amino-acid sequences from human genomic and human, rat and mouse lysyl oxidase cDNAs were subjected to computer comparison. This revealed a highly-conserved primary structure and similar computer-predicted secondary structures. A prototypical lysyl oxidase structural model was reconciled with the known physical, chemical and biological properties. Analysis of the post-translationally-modified and proteolytically-processed mature enzyme model revealed a copper coordination complex that may be contained as part of the active site. This integral copper coordination complex resembles a talon. The proposed model should facilitate the elucidation of these and other structural and functional relationships within the lysyl oxidase molecule.

4-Hydroxycoumarins↗
PubMed1993

Linkage of human spermatid-specific basic nuclear protein genes. Definition and evolution of the P1-->P2-->TP2 locus.

Protamines and transition proteins are highly basic sperm-specific nuclear proteins that serve to compact the DNA within the condensing spermatid nucleus. It has been proposed that the genes for protamine P1 and P2 and transition protein 2 may be clustered at a single loci. This may have marked implications for their evolution and coordinate developmental regulation. The cloning of a contiguous segment encompassing this region would directly address these issues. We have utilized a cloned segment of the human protamine P1 gene to isolate a human cosmid clone which contains the genes for protamines P1 and P2 and transition protein TP2. Sequence and hybridization analysis of this cosmid showed that the TP2 gene resided 3' of the P2 gene at a distance of approximately 7 kilobases (kb). This establishes the order of this gene cluster within a 13-kb region of 16p13.13-16p13.2 to be P1-->P2-->TP2. Similar to their spatial order, comparative computer-assisted sequence analysis predicts an identical relative rank order of emergence. Accordingly we propose that this gene cluster may have arisen from the P1 gene through a series of gene duplication events.

Amino Acid Sequence↗
PubMed1993

Expression and accumulation of lysyl oxidase, elastin, and type I procollagen in human Menkes and mottled mouse fibroblasts.

Menkes syndrome in humans is an X-linked disorder characterized in part by abnormal copper transport, cellular copper sequestration, and defective crosslinking of collagen and elastin. A decrease in the functional activity of lysyl oxidase, a cuproenzyme, is thought in part to be responsible for the decreased crosslinking of collagen and elastin. It has also been suggested that low levels of lysyl oxidase activity may occur secondarily to disturbances in intracellular copper translocation and consequently impaired incorporation of copper into lysyl oxidase. Herein, we examine the expression and accumulation of selected extracellular matrix proteins in fibroblasts from a Menkes patient, as well as fibroblasts from the tortoiseshell (MoTo/y) mouse. The MoTo mutation is an allele of the mottled (Mo) locus, which is considered to be a murine analog of the human Menkes locus. In both Menkes and tortoiseshell fibroblasts, levels of lysyl oxidase mRNA transcripts were less than 15% of levels for corresponding controls. The level of elastin mRNA transcripts was also markedly lower in both cell lines in comparison to controls. In contrast, the levels of procollagen Type I mRNA were similar or enhanced in Menkes and MoTo/y fibroblasts compared to their respective controls. Consequently, we conclude that the connective tissue defects associated with Menkes syndrome and those occurring in mottled mouse mutants involve more than abnormal copper utilization in the formation of lysyl oxidase holoenzyme. Based on the present studies in cell culture, the production of essential enzymes and matrix proteins, such as lysyl oxidase and elastin, appear to be altered at the level of transcription or mRNA turnover.

Animals↗
PubMed1993

The mouse lysyl oxidase gene (Lox) resides on chromosome 18.

Lysyl oxidase initiates crosslink formation of the connective tissue matrix. This enzyme can also revert the ras phenotype in mouse NIH 3T3-transformed cells. Even though lysyl oxidase may participate in many different biological processes, its chromosomal assignment in the mouse genome remains to be addressed. Southern analysis of a panel of Chinese hamster x mouse somatic cell hybrids was utilized to assign the lysyl oxidase gene (Lox) to mouse Chromosome 18.

Animals↗
PubMed1993

Analysis of the core histone gene cluster of the annelid Platynereis dumerilii.

The arrangement of the polychaete annelid Platynereis dumerilii core histone gene cluster and nucleotide sequence has been reported (D. Sellos, S. A. Krawetz and G. H. Dixon, 1990). The H2B and H3 mRNAs are transcribed from one DNA strand, while the H2A and H4 histone mRNAs are transcribed from the other. The H1 gene is not contained as a member of this cluster. Computer assisted sequence analysis of this region was undertaken to define the organization and representation of the various sequence motifs embedded within this region. The analysis revealed that two large regions on opposite strands of the cluster were similar to one another. This organization is reminiscent of an ancient gene duplication event from which the various members independently evolved.

Animals↗
PubMed1993

Gene assignment by quantitative hybridization analysis of somatic cell hybrids.

Since the inception of somatic cell hybridization technology, the number of genes mapped to a particular chromosome or region of a chromosome has increased exponentially. Conventional assignment relies on the interpretation and designation of concordance to the various panel members. Assignment of genes to individual chromosomes may be ambiguous if the representation of the individual chromosomes within the hybrid panel is not considered. To overcome this inherent limitation, we have developed a computer-assisted method to assign genes to chromosomes. This assignment utilizes an integrative statistical analysis procedure to reconcile chromosomal representation of each member of the somatic cell hybrid panel. In this manner, the intensity of the corresponding bands appearing on the autoradiographic image reflects the prevalence of that specific gene-containing chromosome within the somatic cell hybrid. The statistical method described above provides the foundation for an independent means to assign genes to a specific chromosome. We have utilized this method to assign the human lysyl oxidase gene to chromosome 5.

Animals↗
PubMed1992

Purification of cloned and genomic DNA by guanidine thiocyanate/isobutyl alcohol fractionation.

We have developed a rapid and efficient method to purify cloned or high molecular weight eukaryotic DNA from cell lysates using guanidine thiocyanate and isobutyl alcohol. This simple two-step extraction procedure utilizes isobutyl alcohol to fractionate DNA away from cell lysates containing guanidine thiocyanate, yielding a relatively large quantity of pure DNA. The yield of DNA is approximately twice that obtained by the organic extraction method. DNA purified with this chaotropic-based protocol is suitable for a variety of applications including restriction analysis, double-stranded sequencing, PCR, subcloning, and transcription.

Butanols↗
PubMed1992

Characterization of the human lysyl oxidase gene locus.

Lysyl oxidase (EC 1.4.3.13) is a copper-dependent enzyme acting principally on collagen and elastin catalyzing the formation of aldehyde cross-links. It is also believed to possess a tumor suppressor activity as the anti-oncogene of ras. While rat, human, and mouse lysyl oxidase cDNAs have been cloned, little is known about the structure of the gene, its organization, or regulation. This paper describes the cloning of an intronic segment of the human lysyl oxidase gene. Sequence analysis defined the location of an intron that separates the prepro-coding segments from the segment encoding the catalytic domain. Genomic restriction mapping and gene copy number data established that multiple lysyl oxidase mRNA transcripts originate from a single gene and thus are products of alternative splicing. Northern analysis of adult and fetal fibroblast RNA showed a dominant approximately 4.3-kilobase lysyl oxidase mRNA transcript that varied in abundance as a function of cell line. These data are consistent with a complex mechanism regulating the expression of the lysyl oxidase gene.

Adult↗
PubMed1991

Evidence for the evolutionary origin of human chromosome 21 from comparative gene mapping in the cow and mouse.

To determine the extent of conservation between bovine syntenic group U10, human chromosome 21 (HSA 21), and mouse chromosome 16 (MMU 16), 11 genes were physically mapped by segregation analysis in a bovine-hamster hybrid somatic cell panel. The genes chosen for study span MMU 16 and represent virtually the entire q arm of HSA 21. Because the somatostatin gene (SST), an HSA 3/MMU 16 locus, was previously shown to be in U10, the transferrin gene (TF), an HSA 3/MMU 9 marker, was also mapped to determine whether U10 contains any HSA 3 genes not represented on MMU 16. With the exception of the protamine gene PRM1 (HSA 16/MMU 16), all of the genes studied were syntenic on bovine U10. Thus, all homologous loci from HSA 21 that have been studied in the cow are on a single chromosome. The bovine homolog of HSA 21 also carries several HSA 3 genes, two of which have homologous loci on MMU 16. The syntenic association of genes from the q arm of HSA 3 with HSA 21 genes in two mammalian species, the mouse and the cow, indicates that HSA 21 may have that contained genes now residing on HSA 3. Additionally, the syntenic association of TF with SST in the cow permits the prediction that the rhodopsin gene (RHO) is proximal to TF on HSA 3q.

Animals↗
PubMed1991

Exhibition of specific alterations in activities and mRNA levels of rat islet glycolytic and mitochondrial enzymes in three different in vitro model systems for attenuated insulin release.

We studied the possible relationships between the functional status of the beta-cell and activities or mRNA contents of enzymes involved in the catabolism of glucose. Three different in vitro models with attenuated insulin response were used: rat islets cultured at a low glucose concentration, rat islets incubated in vitro with streptozocin, and fetal rat islets. The fetal and streptozocin-administered islets were compared with adult islets cultured in RPMI-1640 containing 11 mM glucose, and the effects of the in vitro glucose concentrations (3.3, 11, and 28 mM) were assessed on adult islets only. Cellular mRNA levels for the mitochondrial DNA-encoded cytochrome b and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by Northern-blot analysis. Enzymatic activities of high-Km (glucokinase) and low-Km (hexokinase) glucose-phosphorylating enzymes and succinate-cytochrome c reductase were also determined. Islets cultured at 3.3 mM glucose displayed a decreased activity of glucokinase compared with islets cultured at 28 mM glucose (23.3 +/- 12%), whereas there was no difference in hexokinase activity or the level of GAPDH mRNA. The activity of succinate-cytochrome c reductase was similar in islets cultured at the different glucose concentrations. The level of cytochrome b mRNA increased at 28 mM glucose compared with islets cultured at 11 mM glucose (140 +/- 14%). Islets incubated with streptozocin and subsequently cultured for 7 days at 11 mM glucose exhibited a decreased level of cytochrome b mRNA (65 +/- 5%) and no differences in the activities of glucokinase, hexokinase, succinate-cytochrome c reductase, or the level of GAPDH mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Organization and complete nucleotide sequence of the core-histone-gene cluster of the annelid Platynereis dumerilii.

The arrangement of the core-histone genes, their transcriptional polarity and their nucleotide sequences have been determined for the polychaete annelid Platynereis dumerili. A clone containing the core-histone genes was isolated from a annelid genomic library constructed in the EMBL-4 phage vector, using a trout H3 genomic probe. This clone was found to contain two and a half repeats of a 6-kbp EcoRV fragment that contained one copy of each of the core-histone genes. The clusters are tandemly arrayed in the genome and the gene order within the core-histone cluster does not vary. Absolutely no differences were found in the nucleotide sequences comprising the same part of two adjacent clusters (bases -225 to 2776 and bases 5821 to 8825). The number of copies of the cluster appeared to be high: approximately 660 copies/diploid cell, as also observed in sea urchins and amphibians. There are also some additional subtypes of histone gene organization: multimers of tandemly arrayed genes and isolated genes; these are present at a much lower copy number (an average of 40-50 copies/diploid genome). Two mRNAs (for H2B and H3) are transcribed from one DNA strand and the two other histone mRNAs (for H2A and H4) from the other strand as is the case for some insects and certain vertebrates. No H1-coding sequence has been found in the completely sequenced four-membered cluster. The organization of histone genes in P. dumerilii is similar to the clustering found in Caenorhabditis elegans but in this nematode worm several different types of organization are observed with a low copy number for each.

Amino Acid Sequence↗
PubMed1990

In situ localization of a mammalian protamine gene: parameters affecting specificity of hybridization.

Southern hybridization analysis of Indian muntjac genomic DNA with the Eco-Taq bovine genomic protamine probe revealed a simple banding pattern. The pattern of hybridization was identical with that previously observed in the genus Bos. This suggested that the bovine probe specifically hybridized to the Indian muntjac protamine gene. The opportunity was thus provided to assign the chromosomal location of the protamine gene in a comparatively simple system. Accordingly, this probe and the corresponding cDNA probe were used for in situ chromosome hybridization and localization. Various parameters affecting specificity and the resolution of hybridization were examined. Subsequent to optimization, the Indian muntjac gene was shown to be autosomal and distally located in the telomeric region of the p arm of chromosome 1.

Animals↗
PubMed1990

Parameters affecting hybridization of nucleic acids blotted onto nylon or nitrocellulose membranes.

Nine different nylon and nitrocellulose membranes were compared utilizing four different methods of attaching the nucleic acid target. Nylon membranes repeatedly demonstrated increased sensitivity as compared to nitrocellulose membranes. Sensitivity could also be enhanced by mildly denaturing the target prior to attachment onto the membrane. This was achieved by either UV cross-linking or baking.

Collodion↗
PubMed1989

Phagemid VPCS vectors for priming, cloning and sequencing.

A phagemid was adapted for use as the vector in the vector-primer-cloner-sequencer cloning system. The use of this new vector markedly expanded the utility of this technology for the construction of cDNA libraries. Technological advantages and new capabilities include: (1) a greater number of unique restriction sites within the polylinker region; (2) the ability to produce single-stranded templates for nucleotide sequencing, and (3) a convenient means to synthesize strand-specific hybridization probes. With the use of this cloning system, a rat liver cDNA library (8.56 x 10(5) recombinants from 1 microgram of poly(A)+ RNA) was rapidly (in two days) constructed.

Cloning, Molecular↗
PubMed1989

Sequence errors described in GenBank: a means to determine the accuracy of DNA sequence interpretation.

The accuracy of nucleic acid sequence data interpretation was determined by assessing and quantifying the discrepancies reported in the GenBank database. This permitted the calculation of an Error Rate (ER) for nucleic acid sequence determination. If one assumes that most entries (TB, Total Bases) were independently verified or those without reported discrepancies were correct, the ER is 0.368 errors per 1000 bases. However, if one assumes that only those sequences with reported discrepancies (TBIQ, Total Bases from entries In Question) were verified and are thus correct, the ER is 2.887 errors per 1000 bases. This establishes the first set of limit boundaries of the ER for sequence interpretation and sequence errors within the GenBank database and provides the foundation for future assessments and the monitoring of sequence data accumulation. In addition, the ER measure provides a basis to evaluate the efficiency and merit of present and future automated nucleic acid sequencing technologies which will have a direct impact upon the final outcome of the "Human Genome Initiative".

Base Sequence↗
PubMed1989

Chromosomal localization and structure of the human P1 protamine gene.

The human P1 protamine gene and mRNA were amplified with the use of the polymerase chain reaction and cloned into PTZ19R. The sequences were determined which revealed the presence of an intron. Southern and Northern hybridization analyses showed that the gene was single copy and that the mRNA was approximately 450 bases long. The gene was mapped to chromosome 16 with the use of a somatic cell hybrid panel and localized to the 21 region of the q arm by in situ hybridization of the human P1 protamine probe to human metaphase chromosomes.

Amino Acid Sequence↗
PubMed1989

Molecular probes for general testicular and specific spermatogenic function.

Northern analysis of human testis poly(A+) RNA with a mixture of oligonucleotide primer extended cDNA probes revealed several similar RNAs. These RNAs were subsequently cloned into a VPCS (vector-primer-cloner-sequencer) plasmid. One of these clones, NDHu1, was represented within the library a number of times and hybridized strongly to a poly(A+) RNA of congruent to 1.2 kb. Sequence analysis identified this clone as the URF 1 subunit of the mitochondrial NADH dehydrogenase (NDHu1). Comparison of the relative levels of the NDHu1 and human protamine 1 (HP1) transcripts revealed that HP1 was less abundant than NDHu1. This was unexpected, since it is known that within differentiating mammalian spermatid cells, protamine (HP1) is an abundant transcript. This suggested that the ratio of the relative levels of these two very different mRNAs was indicative of the relationship between specific spermatogenic function (germ cell transcription, determined by the level of the HP1 transcript) and general testicular cell function (determined by the level of the mitochondrial mRNAs, i.e. NDHu1). This correlation was maintained when several individuals expressing various degrees of testicular dysfunction were examined. This study suggests that these probes may be useful markers for general testicular and specific spermatogenic function.

Base Sequence↗