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S A Krawetz

Publications and source records attributed to S A Krawetz.

At least 37 records · Page 2Linked to original sources
PubMed1999

Reprogramming the male gamete genome: a window to successful gene therapy.

Hematopoiesis and spermatogenesis both initiate from a stem cell capable of renewal and differentiation. Each pathway reflects the expression of unique combinations of facultative, i.e. tissue-specific and constitutive, i.e. housekeeping, genes in each cell type. In spermatogenesis, as in hematopoiesis, commitment is mediated by the mechanism of potentiation whereby specific chromatin domains are selectively opened along each chromosome. Within each open chromatin domain, a unique battery of gene(s) is availed to tissue-specific and ubiquitous transacting factors that are necessary to initiate transcription. In the absence of an open domain, trans-factor access is denied, and the initiation of transcription cannot proceed. Cell-fate is thus ultimately defined by the unique series of open-potentiated cell-specific chromatin domains. Defining the mechanism that opens chromatin domains is fundamental in understanding how differentiation from stem cells is controlled and whether cell-fate can be modified. A recent examination of the mammalian spermatogenic pathway [Kramer, J.A., McCarrey, J.M, Djakiew, D., Krawetz, S.A., 1998. Differentiation: the selective potentiation of chromatin domains. Development 125, 4749-4755] supports the view that cell fate is mediated by global changes in chromatin conformation. This stride underscores the possibility of moderating differentiation through chromatin conformation. It is likely that gene therapeutics capable of selectively potentiating individual genic domains in populations of differentiating and/or replicating cells that modify cellular phenotype will be developed in the next millennium.

Animals↗
PubMed1999

Temporal expression of the transgenic human protamine gene cluster.

OBJECTIVE: To ascertain the fidelity of expression of the genes from the transgenic human sperm-specific nuclear packaging protamine-1-->protamine-2-->transition protein-2 (PRM1-->PRM2-->TNP2) locus. DESIGN: Controlled human transgene study. SETTING: Basic science laboratory. ANIMAL(S): Age-matched transgenic and nontransgenic mice. INTERVENTION(S): Transgenic mice containing the human protamine locus were mated. One testis from each offspring was frozen at -80 degrees C and the other was preserved in formalin. MAIN OUTCOME MEASURE(S): The temporal expression of the human and mouse protamines was evaluated by Northern blot analysis. Orientation of the transgenic locus was determined by Southern blot analysis. Tissue morphology was assessed histologically. RESULT(S): Conservation of transgenic morphology was confirmed. Head-to-tail integration of the PRM1--> PRM2-->TNP2 locus was shown. Temporal expression of the mouse and human protamine genes was maintained in the transgenic state. CONCLUSION(S): These results show that the head-to-tail concatomer of the PRMI-->PRM2-->TNP2 locus contains all the necessary elements for appropriate temporal expression while maintaining testicular structure and function.

Aging↗
PubMed1999

Lysyl oxidase transcripts in peritoneal adhesions and incisional scars.

OBJECTIVE: To evaluate the role of lysyl oxidase in postsurgical adhesion formation and incision wound repair. STUDY DESIGN: Female New Zealand rabbits underwent a pelvic-peritoneum adhesion-inducing operation under sterile conditions. In brief, the uterine horns were removed from the abdomen and abraded with surgical gauze and a scalpel blade. The horns were then replaced into the abdominal cavity, the incision was sutured, and the animals were allowed to recover. The animals were killed before lesion development and after 2, 4, 8, and 14 days of postsurgical recovery. The abraded uterine horns, abdominal wall incisional wound and a portion of the sidewall peritoneum were then removed. Total RNA was extracted using the guanidinium thiocyanate-phenol-chloroform method. Northern blot analysis was performed with an [alpha-32P]-labeled lysyl oxidase probe. RESULTS: Lysyl oxidase was expressed during abdominal wall incision repair on days 2 and 4 of postsurgical recovery, declining thereafter (days 8 and 14). In contrast, no increase in lysyl oxidase expression was noted in the uterine horns as compared to the control sidewall peritoneum. CONCLUSION: Lysyl oxidase plays a differential role in the early stages of abdominal wall and uterine horn repair.

Animals↗
PubMed1998

MARs mission update.

Genes are organized as chromatin domains and positioned in the nucleus through regions of nuclear matrix association, termed MARs. Although well-studied, the mechanisms regulating expression of the beta-globin locus remain an enigma. Here, we show that certain MAR sequences of the beta-globin locus are conserved and reiterated throughout the genome in association with other genes of the hematopoietic lineage. Further, the density of the MARs within the beta-globin locus and the sharing of these sequences by the various members of this gene cluster suggest that they may provide critical gene regulatory components.

Base Sequence↗
PubMed1998

Genesis of a novel human sequence from the protamine PRM1 gene.

The members of the male haploid expressed protamine 1 (PRM1)-->protamine 2 (PRM2)-->transition protein 2 (TNP2) locus exist as a single, coordinately expressed genic domain. Previous analysis has revealed that the genes within the human PRM1-->PRM2-->TNP2 domain are inter-related, as they share significant sequence similarity at both the nucleotide and amino acid levels. Analysis described here supports the view that a fourth candidate coding region, gene4/Prm3, was derived from PRM1 during the genesis of the PRM1-->PRM2-->TNP2 domain. In some species, gene4 has diverged to a great extent, which can limit its expression.

Animals↗
PubMed1998

A matrix associated region localizes the human SOCS-1 gene to chromosome 16p13.13.

The MarFinder algorithm was applied to a newly sequenced segment of 16p13.13 abutting the 3' end of the human PRM1-->PRM2-->TNP2 locus. A candidate region of matrix attached was identified. Subsequent biophysical analysis showed that this region was attached to the somatic nuclear matrix. Nucleotide sequence analysis also revealed the presence of a CpG island. Data base queries showed that this region contained the SOCS-1 gene. Thus, the SOCS-1 gene is bounded by a somatic MAR and is just 3' of the spermatid-expressed PRM1-->PRM2-->TNP2 domain at position 16p13.13.

Carrier Proteins↗
PubMed1998

Centromere sequences localize to the nuclear halo of human spermatozoa.

Chromatin is organized into a series of discrete nuclear matrix-associated and non-nuclear matrix-associated domains. The non-matrix-associated domains consist of loops of DNA that are attached to the proteinaceous nuclear matrix by matrix-associated regions (MARs). Although this organization is well characterized in somatic cells, comparatively little is known of this mode of organizing the genome in the human sperm nucleus. To define this relationship, the interaction of human sperm chromatin with the nuclear matrix was assessed by fluorescence in situ hybridization using specific alpha satellite probes directed to the centromeric regions of chromosomes 13 plus 21 and 18. Hybridization of the centromeric sequences was visualized as segmented, bundled structures that extended from the nuclear core into the halo.

Cell Nucleus↗
PubMed1998

Differentiation: the selective potentiation of chromatin domains.

Potentiation is requisite for the expression of our genome. It is the mechanism of opening chromatin domains to render genes accessible to tissue-specific and ubiquitous transacting-factors that enables transcription. The results presented in this study demonstrate that modulation of stage- and cell-type-specific gene expression during mammalian spermatogenesis involves selective potentiation of testis-expressed genes that reverses their repressive state when present in the spermatogonial stem cell. This directly contrasts hematopoiesis, which acts to selectively restrict lineage potential during differentiation from its permissive stem cell. These results are key to understanding how differentiative pathways are controlled and cellular phenotypes determined. A window to their modulation is presented.

Aging↗
PubMed1997

Mathematical model to predict regions of chromatin attachment to the nuclear matrix.

The potentiation and subsequent initiation of transcription are complex biological phenomena. The region of attachment of the chromatin fiber to the nuclear matrix, known as the matrix attachment region or scaffold attachment region (MAR or SAR), are thought to be requisite for the transcriptional regulation of the eukaryotic genome. As expressed sequences should be contained in these regions, it becomes significant to answer the following question: can these regions be identified from the primary sequence data alone and subsequently used as markers for expressed sequences? This paper represents an effort toward achieving this goal and describes a mathematical model for the detection of MARs. The location of matrix associated regions has been linked to a variety of sequence patterns. Consequently, a list of these patterns is compiled and represented as a set of decision rules using an AND-OR formulation. The DNA sequence was then searched for the presence of these patterns and a statistical significance was associated with the frequency of occurrence of the various patterns. Subsequently, a mathematical potential value,MAR-Potential, was assigned to a sequence region as the inverse proportion to the probability that the observed pattern population occurred at random. Such a MAR detection process was applied to the analysis of a variety of known MAR containing sequences. Regions of matrix association predicted by the software essentially correspond to those determined experimentally. The human T-cell receptor and the DNA sequence from the Drosophila bithorax region were also analyzed. This demonstrates the usefulness of the approach described as a means to direct experimental resources.

Base Sequence↗
PubMed1997

Ethanol-induced intracellular calcium mobilization rapidly alters gene expression in the mouse blastocyst.

The induction of intracellular Ca2+ release in pre-implantation mouse embryos accelerates their subsequent rate of development in vitro through a calmodulin-dependent mechanism [Stachecki J.J., Armant D.R. Transient release of calcium from inositol 1,4,5-trisphosphate-specific stores regulates mouse pre-implantation development. Development 1996; 122: 2485-2496]. To examine the hypothesis that intracellular Ca2+ signaling alters embryonic gene expression, individual transcript levels were compared by mRNA differential display before and 1 h after intracellular Ca2+ mobilization with ethanol in mouse blastocysts. Ten up-regulated and four down-regulated genes were observed, representing 3.5% of approximately 400 transcripts that were resolved. After sequencing, most of the DNA fragments appeared to be novel; however, two amplicons that increased after Ca2+ mobilization were identified as arginase and ubiquitin conjugating enzyme (E2). The up-regulation of arginase mRNA (3.5-fold after 2 h) was confirmed by reverse transcription and the polymerase chain reaction using specific oligonucleotide primers derived from the deduced mouse embryo sequence. A corresponding 2.5-fold increase in arginase enzymatic activity peaked 9 h after ethanol exposure. Increased expression of arginase and other genes may mediate the onset of rapid cell proliferation and differentiation that is induced by Ca2+ signaling during pre-implantation development.

Animals↗
PubMed1997

The use of cloned repetitive sequences as hybridization competitors to detect single copy sequences.

To suppress background hybridization due to repetitive sequence elements, competitor Alu containing clones were isolated from a subclone library of human cosmid clone hP3.1. Pre-annealing of the probe-with this competitor increased the signal well above background. In comparison, the addition of the competitor directly to both the prehybridization and hybridization solution was more effective in reducing background. This dramatically increased the signal to noise ratio of the specific hybridization event. Application to fluorescent in situ hybridization (FISH) is readily apparent.

Binding, Competitive↗
PubMed1997

Lysyl oxidase, cellular senescence and tumor suppression.

Replicative senescence may provide a mechanism of tumor suppression and tumor suppressor genes of the extracellular matrix, like lysyl oxidase, may play a role in cellular senescence. To test this hypothesis and determine whether the extracellular matrix may serve as a marker, the steady-state levels of human lysyl oxidase, alpha-I type III collagen and beta-actin transcripts were assessed in various cell lines during in vitro passage. Northern hybridization analysis showed a significant increase in the levels of progeria fibroblast extracellular matrix mRNAs immediately preceding senescence. The levels of these mRNAs were unaffected in age-matched normal fibroblast and fetal fibroblast cell lines.

Actins↗
PubMed1997

Genetic testing for male infertility: a postulated role for mutations in sperm nuclear matrix attachment regions.

Numerous reports have suggested that disturbances in nuclear condensation may result in male infertility. This notion has been supported by the observation of infertile individuals with a decrease or absence of the male sperm-specific chromatin packaging protamine proteins. To date, no correlation between the absence of protamine proteins and a mutation within the coding regions of the protamine genes has been documented. To address this issue, PCR-based mutation scanning analysis has been performed across the human male haploid expressed PRM1-->PRM2-->TNP2 domain in several oligozoospermic infertile individuals. This analysis identified a candidate mutation in a region of contact with the sperm nuclear matrix from 2 of 5 affected individuals. This is the first report of a mutation scan covering the entire PRM1-->PRM2-->TNP2 locus in affected individuals.

Chromosomal Proteins, Non-Histone↗
PubMed1997

Haploid transcripts persist in mature human spermatozoa.

Mammalian spermiogenesis is marked by the morphological and functional differentiation of round haploid spermatids into mature spermatozoa. A molecular restructuring of the chromatin accompanies this process facilitated by the transition proteins and protamines which compact and condense the genetic material within the developing spermatid. Previous studies from this laboratory have demonstrated that human protamines PRM1, PRM2 and transition protein TNP2 transcripts are associated with round and elongating spermatids. Extending this investigation, we examined the occurrence of these transcripts in mature spermatozoa by in-situ hybridization analysis using [35S]-labelled cRNA probes. These results demonstrate that PRM1, PRM2 and TNP2 haploid-specific transcripts are present in mature spermatozoa. Quantitative analysis of the localized signal also indicates that the PRM1, PRM2 and TNP2 transcripts persist at a similar ratio to that previously described for these transcripts in human testes, i.e. PRM2 > PRM1 approximately equal to TNP2. The persistence of these transcripts in mature spermatozoa warrants further investigation.

Chromosomal Proteins, Non-Histone↗
PubMed1997

RNA in spermatozoa: implications for the alternative haploid genome.

The presence of specific messenger RNAs in the nuclei of mature mammalian spermatozoa has been demonstrated by several independent laboratories. Others have suggested that various polymerases may also be active in mature spermatozoa. This has led to the notion that the 'sleeping' genome may not be so quiescent after all. The alternate use of somatic-like nucleosomal and haploid protamine packaging structures to assemble sperm chromatin and the ordered array of chromosomes within the mature human sperm nucleus support this view. This had led us to address the issue of whether a somatic-like organization of select regions of the paternal genome and the mRNAs present in spermatozoa were correlated. Results from this and other laboratories suggest that this in indeed the case. Potential roles for this novel packaging and the accumulation of transcripts within the mature human nucleus are discussed.

Animals↗