Phthalates impact on the epigenetic factors contributed specifically by the father at fertilization.

BACKGROUND: Preconception exposure to phthalates such as the anti-androgenic dibutyl-phthalate (DBP) impacts both male and female reproduction, yet how this occurs largely remains unknown. Previously we defined a series of RNAs expressly provided by sperm at fertilization and separately, and in parallel, those that responded to high DBP exposure. Utilizing both populations of RNAs, we now begin to unravel the impact of high-DBP exposure on those RNAs specifically delivered by the father. RESULTS: Enrichment of RNAs altered by DBP exposure within the Molecular Signature Database highlighted cellular stress, cell cycle, apoptosis, DNA damage response, and gene regulation pathways. Overlap within each of these five pathways identified those RNAs that were specifically (≥ fivefold enriched) or primarily (≥ twofold enriched) provided as part of the paternal contribution compared to the oocyte at fertilization. Key RNAs consistently altered by DBP, including CAMTA2 and PSME4, were delivered by sperm reflective of these pathways. The majority (64/103) of overlapping enriched gene sets were related to gene regulation. Many of these RNAs (45 RNAs) corresponded to key interconnected CRREWs (Chromatin remodeler cofactors, RNA interactors, Readers, Erasers, and Writers). Modeling suggests that CUL2, PHF10, and SMARCC1 may coordinate and mechanistically modulate the phthalate response. CONCLUSIONS: Mediated through a CRREW regulatory network, the cell responded to exposure presenting stressed-induced changes in the cell cycle-DNA damage-apoptosis. Interestingly, the majority of these DBP-responsive epigenetic mediators' direct acetylation or deacetylation, impacting the sperm's cargo delivered at fertilization and that of the embryo.

Cleavage of rRNA ensures translational cessation in sperm at fertilization.

Intact ribosomal RNAs (rRNAs) comprise the majority of somatic transcripts, yet appear conspicuously absent in spermatozoa, perhaps reflecting cytoplasmic expulsion during spermatogenesis. To discern their fate, total RNA retained in mature spermatozoa from three fertile donors was characterized by Next Generation Sequencing. In all samples, >75% of total sequence reads aligned to rRNAs. The distribution of reads along the length of these transcripts exhibited a high degree of non-uniformity that was reiterated between donors. The coverage of sequencing reads was inversely correlated with guanine-cytosine (GC)-richness such that sequences greater than ∼70% GC were virtually absent in all sperm RNA samples. To confirm the loss of sequence, the relative abundance of specific regions of the 28S transcripts in sperm was established by 7-Deaza-2'-deoxy-guanosine-5'-triphosphate RT-PCR. The inability to amplify specific regions of the 28S sequence from sperm despite the abundant representation of this transcript in the sequencing libraries demonstrates that approximately three-quarters of RNA retained in the mature male gamete are products of rRNA fragmentation. Hence, cleavage (not expulsion of the RNA component of the translational machinery) is responsible for preventing spurious translation following spermiogenesis. These results highlight the potential importance of those transcripts, including many mRNAs, which evade fragmentation and remain intact when sperm are delivered at fertilization. Sequencing data are deposited in GEO as: GSE29160.

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.

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.

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)

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.

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.

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.

A complex population of RNAs exists in human ejaculate spermatozoa: implications for understanding molecular aspects of spermiogenesis.

The presence of mRNAs in human ejaculate spermatozoa is well established, yet little is known of the representation or function of these transcripts. To address these issues, the complexity of spermatozoal RNA was examined. As expected, testis-expressed mRNAs were detected by RT-PCR in mature human spermatozoa. Interestingly, when a testis cDNA library was probed with total spermatozoal RNA, less than 2% of plaques gave a strong hybridization signal, suggesting a rather unique sperm-derived population. To further define the sequence distribution, 18 strongly hybridizing clones were selected at random for end-sequence analysis. Twelve matched unique sequences in the EST, STS and NR databases, whereas five showed no similarity to any of the sequences in the databases. In addition, one clone belonged to the SINE repetitive element family. As demonstrated by sequencing randomly primed cloned inserts, short (SINE/MER) or long (LINE/ORF2) interspersed repeat-like sequences are also contained as part of the spermatozoal RNA fraction. It is now evident that human spermatozoa contain a rich repertoire of both known and unknown protein-encoding and non-coding RNAs. This provides a unique opportunity to identify and investigate the many genes responsible for the structure and function/dysfunction of the male gamete using spermatozoal RNA as the template.

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.

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.

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.

Recovering filter-based microarray data for pathways analysis using a multipoint alignment strategy.

The use of commercial microarrays is rapidly becoming the method of choice for profiling gene expression and assessing various disease states. Research Genetics has provided a series of biological and software tools to the research community for these analyses. The fidelity of data analysis using these tools is dependent on a series of well-defined reference control points in the array. During the course of our investigations, it became apparent that in some instances the reference control points that are required for analysis became lost in background noise. This effectively halted the analysis and the recovery of any information contained within that experiment. To recover this data and to increase analytical veracity, the simple strategy of superimposing a template of reference control points onto the experimental array was developed. The utility of this tool is established in this communication.

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.

Characterizing a human lysyl oxidase chromosomal domain.

The expression of each locus in our genome is regulated by a gene-potentiative mechanism, whereby the gene first assumes the necessary structural conformation to enable transcription. This serves as the corner-stone for the three-tiered regulatory mechanism of potentiation, i.e., the opening of a chromatin domain, initiation of transcription, and transcript elongation. Although this is now generally accepted as the pathway that mediates gene expression, it has never been shown directly to control the expression of any heart-related gene. Lysyl oxidase enzymatically crosslinks members of the extracellular matrix, including elastin and collagen. Formation of these structures is essential to development and tissue repair. This system has enabled us to begin to address the underlying mechanism governing the selection of connective tissue genes for expression. However, before one can dissect this mechanism, it is necessary to define and characterize the locus, i.e., the corresponding genic domain. Our progress toward creating the resources necessary to unravel this mechanism is summarized in this review.

Design and implementation of an introductory course for computer applications in molecular genetics. A case study.

Formal training in computational biology was initiated at Wayne State University in 1990 to meet the needs of the faculty. This was still at a time when the molecular databases and analysis tools could be housed in what is now equivalent to a modern but dated desktop computer. In 1995 the course was expanded to include graduate students to provide these senior students with a foundation in computational biology. This course has armed our students with a requisite set of basic skills that are necessary for a successful career in molecular genetics. It is now an integral component of the graduate program of the Center for Molecular Medicine and Genetics and our experiences in course delivery have been detailed (BioInformatics Methods and Protocols, S. Misener and S. A. Krawetz, eds., Humana Press, Totowa, NJ, 2000.). The course was expanded to a campus-wide unlimited enrollment program for the summer of 2000 to address the needs of our student body. In this review we present our experience with delivering a multidisciplinary campus-wide computational biology course to a new and widely diverse student body.

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.

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.

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.

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.