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S A Krawetz

Publications and source records attributed to S A Krawetz.

At least 55 records · Page 3Linked to original sources
PubMed1996

Nuclear matrix interactions within the sperm genome.

Analysis of the haploid-expressed human PRM1 --> PRM2 --> TNP2 genic domain has revealed two regions of attachment to the sperm nuclear matrix. These sperm nuclear matrix attachment regions delimit the DNase I-sensitive domain of this haploid-expressed locus. The domain is intermediately associated with but not attached to the nuclear matrix. DNase I-sensitive genes within the mature sperm nucleus, such as protamine 1, protamine 2, transition protein 2, alpha-globin, and beta-actin, display this intermediate affinity for the sperm nuclear matrix. This may denote their role in templating the male genome prior to fertilization, thus ensuring the formation of a viable male pronucleus during early embryonic development.

Base Sequence↗
PubMed1996

Computer-assisted search for sites of nuclear matrix attachment.

This communication describes a useful approach for assessing the significance of the occurrence of multiple motifs within defined segments of the genome. Regions of potential biological importance are identified using various sequence motifs, and then the results are displayed as their cumulative statistically weighted distribution. We illustrate the utility of this strategy to the search for known nuclear matrix-associated regions by its application to the human beta-globin and other loci.

Algorithms↗
PubMed1995

A haploid expressed gene cluster exists as a single chromatin domain in human sperm.

Mammalian spermiogenesis is marked by the initial disruption of the nuclear-histone-DNA complex by the transition proteins for ultimate replacement with protamines. The genes for three of these low molecular weight basic nuclear proteins exist as a single linear array of PRM1, PRM2, and TNP2 on human chromosome 16p13.2. To begin to address the mechanism governing their transcriptional potentiation, a region of approximately 40 kilo-bases of the human genome encompassing these genes was introduced into the germ line of mice. Fluorescence in situ hybridization and Southern analysis showed that this segment of the human genome integrated into independent chromosomal sites while maintaining its fidelity. Transcript analysis demonstrated that the expression of the endogenous mouse protamine Prm1 and Prm2 genes as well as the mouse transition protein Tnp2 gene were expressed along with their human transgene counterparts. The pattern of expression of these transgenic human genes within this multigenic cluster faithfully represented that observed in vivo. In addition, all members of this transgenic gene cluster were expressed in proportions similar to those in human testis. Copy number-dependent and position-independent expression of the transgenic construct demonstrated that the corresponding biological locus was contained within this segment of the human genome. Furthermore, DNase I sensitivity established that in sperm the human PRM1-->PRM2-->TNP2 genic domain was contained as an approximately 28.5-kilobase contiguous segment bounded by an array of nuclear matrix associated topoisomerase II consensus sites. This is the first description of a multigenic male gamete-specific domain as a fundamental gene regulatory unit. A model of haploid-specific gene determination is presented.

Amino Acid Sequence↗
PubMed1995

CLONEPLACER: a software tool for simulating contig formation for ordered shotgun sequencing.

This communication describes a software tool that enables one to simulate large-scale regional mapping using an ordered shotgun sequencing approach. The analysis routines that are provided yield an estimate of the depth of coverage of the physical map, the largest contig formed, and the number of gaps remaining at any given juncture in the project. A detailed listing describing the span of each contig within the physical map is also presented. This provides an a priori means of estimating the resources that will be required to undertake any megabase mapping or sequencing project. CLONEPLACER provides the much needed guide to deriving the optimal strategy.

Chromosome Mapping↗
PubMed1995

Rapid inhibition of mRNA synthesis during preimplantation embryo development: vital permeabilization by lysolecithin potentiates the action of alpha-amanitin.

Lysolecithin was used to permeabilize embryonic cells to impermeant compounds without compromising embryo viability. Within 2 min of exposure to 0.05% lysolecithin, mouse morulae became permeable to inositol 1,4,5-trisphosphate, leading to the release of calcium from intracellular stores. Although these morulae remained permeable for at least 2 h, they developed normally to the blastocyst stage during subsequent culture in vitro. Lysolecithin permeabilization was then used to potentiate the internalization of alpha-amanitin. Pretreatment with lysolecithin for 2 min markedly accelerated the onset of transcriptional inhibition from 3 h to 10 min after alpha-amanitin addition. The rapid inhibition by alpha-amanitin of mRNA synthesis in lysolecithin-treated embryos provided a precise method for delineating developmentally important transcriptional periods. Using this protocol, we found that mRNAs required for embryonic cavitation were synthesized between 87 and 91 h post-hCG, shortly before blastocoel formation commenced.

Amanitins↗
PubMed1995

Isolation and characterization of a 1 Mb region of 5q23.3-q31.2 surrounding the human lysyl oxidase gene.

Lysyl oxidase (EC 1.4.3.13) plays a pivotal role in the maintenance of tissue integrity in both the normal and pathological states. It is a member of a newly discovered gene family that exhibits a complex mode of regulation. To date the resources necessary to begin to address its regulation have not been assembled. In part, this reflects the instability of this region of the genome when cloned into cosmid vectors. The paucity of long range restriction endonuclease sites suitable for mapping this region of the genome has further hampered progress. To begin to address this issue 2 YAC clones of 920 kb and 245 kb that contain the human lysyl oxidase gene were isolated. Long range physical mapping revealed that the 245 kb clone was centrally located within the 920 kb clone. The corresponding map of this region is congruent with that observed in the human genome. Thus, these YACs faithfully represent this region of the human genome. The results of our cloning and mapping studies described in this communication should accelerate the advance of our understanding of this new connective tissue gene family.

Base Sequence↗
PubMed1995

Coordinate expression of the PRM1, PRM2, and TNP2 multigene locus in human testis.

Maintenance of the transcriptionally inert state of the mature human spermatozoon requires the expression of the various members of the human protamine gene cluster prior to the final stages of spermatogenesis. During this process, known as spermiogenesis, round spermatids morphologically differentiate into mature spermatozoa. The expression of the PRM1, PRM2, and TNP2 genes facilitates the compaction and condensation of the genetic material within the developing spermatid. To understand better the coordinate control governing this transformation, we have examined the localization and distribution of the human protamines PRM1 and PRM2 and transition protein TNP2 transcripts during human spermatogenesis. The stage-specific expression of these transcripts was determined by in situ hybridization analysis using [alpha-35S]-labeled cRNA probes. PRM1, PRM2, and TNP2 transcripts were abundant in association with round and elongating spermatids, located in the adluminal region of the seminiferous epithelium. They were not observed in association with spermatogonia, spermatocytes, Sertoli cells, or interstitial cells. These data indicate that the human PRM1, PRM2, and TNP2 transcripts are expressed postmeiotically in round and elongating spermatids. The quantitative evaluation of each transcript was determined as a function of the relative optical density per unit area. In all cases examined, the relative level of each transcript was consistent with the following pattern, PRM2 > PRM1 congruent to TNP2.

Chromosomal Proteins, Non-Histone↗
PubMed1995

DNAView: a quality assessment tool for the visualization of large sequenced regions.

This communication describes DNAView, a graphical tool for the visualization and printing of large nucleic acid sequences. DNAView uses color coding to compactly display genomic segments of up to 100 kb on a single printed page. The specific color schemes integrated into DNAView can highlight 'local aggregate' properties of large segments of DNA. We have also incorporated a confidence expression for the assigned sequence. This is represented by base color intensity that is proportional to the number of times that base was sequenced. Areas of interest, such as exons, introns, repetitive elements and splice sites, can be emphasized using overlays. The colored image can be saved in a standard TIFF image file format that may be imported and annotated by other application software.

Base Sequence↗
PubMed1995

Computer assisted promoter analysis of a human sperm specific nucleoprotein gene cluster.

The promoter regions of the clustered human spermatid-specific nucleoprotein PRM1, PRM2 arid TNP2 genes were compared to define regulatory elements that may govern their expression. Sequence alignment revealed two wll conserved motifs, despite a lack of extensive homology. They are located at similar positions within the first 400 nt of their 5' UTRs. Conservation of these motifs may reflect selective evolutionary pressure to maintain their structure. This supports the view that these elements assume a central role in the coordinate regulation of this gene cluster during spermiogenesis. The distribution of binding sites of known transcription factors was also assessed within the regions flanking the 5' ends of these genes. This analysis should prove useful in directing studies that define the signals necessary for the coordinate regulation of this spermatid specific gene cluster.

Base Sequence↗
PubMed1995

Mapping the clonally unstable recombinogenic PRM1-->PRM2-->TNP2 region of human 16p13.2.

We have isolated a human cosmid clone encompassing approximately 40 kb of the chromosome 16p13.2 region. This region contains the protamine genes PRM1 and PRM2 and the transition protein gene TNP2. To facilitate the characterization of this under represented region of the chromosome 16 physical map, a detailed map of this cosmid clone was constructed. Analysis revealed that this gene cluster was nestled amid numerous repetitive elements. Under representation of this segment may be attributed to these repetitive sequence elements scattered throughout this region rendering this locus clonally unstable. Verification of the map by cosmid and genomic Southern analysis resolved discrepancies within the literature and showed that this highly repetitive DNA sequence containing cosmid was representative of this region of the human genome. This dynamic and recombinogenic region continues to evolve.

Blotting, Southern↗
PubMed1994

Characterization of a human locus in transition.

The spermatid-specific nucleoprotamine genes PRM1 and PRM2 and the transition protein gene TNP2 are clustered at a single site on human chromosome 16p13.2. To begin to understand the mechanism governing their genesis and coordinate regulation the primary sequence of this approximately 40.6 kilobase region was determined. This cluster of genes is embedded within a series of repetitive elements, including numerous Alu elements distributed at a frequency of > 1 Alu element/kilobase. Multiple Alu elements have integrated into separate truncated L1 sequences within this region. Many of these Alu elements are tandemly inserted or clustered. The role of repetitive elements in the genomic organization and evolution of this gene cluster is discussed. Computer-assisted sequence analysis revealed the presence of structural sequence elements often associated with the boundary regions of active transcriptional domains. Further analysis identified a CpG island at the 3' end of this segment of chromosome 16 and other candidate coding segments within this region indicative of an additional linked gene. These sequence landmarks are commensurate with the complexity of the region.

Base Sequence↗
PubMed1994

The origin of lysyl oxidase.

Lysyl oxidase initiates crosslink formation of the collagen and elastin extracellular matrix, thereby delimiting its expansive properties. Recently lysyl oxidase has been cloned from several species enabling the computation of the relative order of appearance of the various components of this enzyme system. Comparative evolutionary computer-assisted sequence analysis of the enzyme and its various substrates was undertaken to address this issue. These results support the ordered genesis of the collagen substrate-->lysyl oxidase enzyme-->elastin substrate.

Animals↗
PubMed1994

ISWAC: proposed system for the integrated assembly of chromosomes.

The generation of a physical map as an integral part of sequence project management is a problem that present computer systems do not address. Primarily, the analysis performed is based solely on the information available from a single knowledge level. Management systems that are currently available do not adequately model the multi-layer top down strategy that is most often utilized to manage large scale sequencing projects. Single layered approaches reflect an algorithmic inadequacy since interacting data sets are required to provide a good solution. The analysis tool that is currently under development termed ISWAC, the Integrated System for Wholistic Assembly of Chromosomes, overcomes these limitations by integrating information available from five layers of knowledge. These knowledge layers utilize information from the linkage map, physical map, restriction map, clone strategy map and the DNA sequence itself. The approach we are implementing, reviews current project status and continually refines the experimental strategy necessary to efficiently complete the sequencing task. To facilitate project completion the system is designed to interactively recommend strategies based on partial information. The utility of this tool is enhanced by implementing knowledge representation techniques that allow reasoning with approximate concepts characteristic of these data-sets. In addition, the raw physical data is maintained within an integrated map database to ease data verification. This paper presents the first discussion of the design specifications for a computer system to assimilate the various forms of data that are being generated as part of the human genome project. It was specifically written to stimulate discussion regarding data standardization, translation, analysis and most important, an understandable user-interphase for the molecular biologist. We would hope that interested readers would respond by assisting in the definition of a set of universal data standards and adopting them in their laboratories.

Algorithms↗