Cyclin B synthesis is required for sea urchin oocyte maturation

Sea urchins are members of a limited group of animals in which meiotic maturation of oocytes is completed prior to fertilization. This is different from oocytes of most animals such as mammals and amphibians in which fertilization reactivates an arrested meiotic cycle. Using a recently developed technique for in vitro maturation of sea urchin oocytes, we analyzed the role of cyclin B, the regulatory component of maturation-promoting factor, in the control of sea urchin oocyte meiotic induction and progression. Oocytes of the sea urchin Lytechinus variegatus accumulate significant amounts of cyclin B mRNA and protein during oogenesis. We analyzed cyclin B synthetic requirements in oocytes and early embryos by inhibiting cyclin B synthesis with DNA and morpholino antisense oligonucleotides. Cyclin B synthesis is not necessary for the entry of G2-arrested oocytes into meiosis; however, it is required for the proper progression through meiotic divisions. Surprisingly, mature sea urchin eggs contain significant cyclin B protein following meiosis that serves as a maternal store for early cleavage divisions. We also find that cyclin A can functionally substitute for cyclin B in early embryos but not in oocytes. These studies provide a foundation for understanding the mechanism of meiotic maturation independent of the zygotic cell cycle

Coordinating cell cycle-regulated histone gene expression through assembly and function of the Histone Locus Body

Metazoan replication-dependent (RD) histone genes encode the only known cellular mRNAs that are not polyadenylated. These mRNAs end instead in a conserved stem-loop, which is formed by an endonucleolytic cleavage of the pre-mRNA. The genes for all 5 histone proteins are clustered in all metazoans and coordinately regulated with high levels of expression during S phase. Production of histone mRNAs occurs in a nuclear body called the Histone Locus Body (HLB), a subdomain of the nucleus defined by a concentration of factors necessary for histone gene transcription and pre-mRNA processing. These factors include the scaffolding protein NPAT, essential for histone gene transcription, and FLASH and U7 snRNP, both essential for histone pre-mRNA processing. Histone gene expression is activated by Cyclin E/Cdk2-mediated phosphorylation of NPAT at the G1-S transition. The concentration of factors within the HLB couples transcription with pre-mRNA processing, enhancing the efficiency of histone mRNA biosynthesis

A Study of the Action of Potassium Hypobromite and Sodium Hypochlorite on Adipamide

The Hoffman reaction of an amide with a hypohalite to give a primary amine in excellent yields is the basis of many syntheses in organic chemistry. This project is concerned with the study of the action of sodium hypobromite and sodium hypochloride on adipamide. Isolation of reaction products, if possible, identification of reaction products, if possible, identification of the products, and determination of the yield of the reaction were desired. It was hope that a diamine, putresecine, would result from this reaction, and therefore the steps in isolation of the products were carried out with this in view

The Histone 3'-Terminal Stem-Loop-Binding Protein Enhances Translation through a Functional and Physical Interaction with Eukaryotic Initiation Factor 4G (eIF4G) and eIF3

Metazoan cell cycle-regulated histone mRNAs are unique cellular mRNAs in that they terminate in a highly conserved stem-loop structure instead of a poly(A) tail. Not only is the stem-loop structure necessary for 3'-end formation but it regulates the stability and translational efficiency of histone mRNAs. The histone stem-loop structure is recognized by the stem-loop-binding protein (SLBP), which is required for the regulation of mRNA processing and turnover. In this study, we show that SLBP is required for the translation of mRNAs containing the histone stem-loop structure. Moreover, we show that the translation of mRNAs ending in the histone stem-loop is stimulated in Saccharomyces cerevisiae cells expressing mammalian SLBP. The translational function of SLBP genetically required eukaryotic initiation factor 4E (eIF4E), eIF4G, and eIF3, and expressed SLBP coisolated with S. cerevisiae initiation factor complexes that bound the 5' cap in a manner dependent on eIF4G and eIF3. Furthermore, eIF4G coimmunoprecipitated with endogenous SLBP in mammalian cell extracts and recombinant SLBP and eIF4G coisolated. These data indicate that SLBP stimulates the translation of histone mRNAs through a functional interaction with both the mRNA stem-loop and the 5' cap that is mediated by eIF4G and eIF3

stringcdc25 and cyclin E are required for patterned histone expression at different stages of Drosophila embryonic development

Metazoan replication-dependent histone mRNAs accumulate to high levels during S phase as a result of an increase in the rate of histone gene transcription, pre-mRNA processing, and mRNA stability at the G1–S transition. However, relatively little is known about the contribution of these processes to histone expression in the cell cycles of early development, which often lack a G1 phase. In post-blastoderm Drosophila embryos, zygotic expression of the stgcdc25 phosphatase in G2 activates cyclin/cdc2 kinases and triggers mitosis. Here we show that histone transcription initiates in late G2 of cycle 14 in response to stgcdc25 and in anticipation of S phase of the next cycle, which occurs immediately following mitosis. Mutation of stgcdc25 arrests cells in G2 and prevents histone transcription. Expression of a mutant form of Cdc2 that bypasses the requirement for stgcdc25 activates histone transcription during G2 in stgcdc25 mutant embryos. Thus, in these embryonic cycles, histone transcription is controlled by the principal G2–M regulators, stringcdc25, and cdc2 kinase, rather than solely by regulators of the G1–S transition. After the introduction of G1–S control midway through embryogenesis, histone expression depends on DNA replication and the function of cyclin E, and no longer requires stgcdc25. Thus, during the altered cell cycles of early animal development, different cell cycle mechanisms are employed to ensure that the production of histones accompanies DNA synthesis

TUT7 catalyzes the uridylation of the 3′ end for rapid degradation of histone mRNA

The replication-dependent histone mRNAs end in a stem–loop instead of the poly(A) tail present at the 3′ end of all other cellular mRNAs. Following processing, the 3′ end of histone mRNAs is trimmed to 3 nucleotides (nt) after the stem–loop, and this length is maintained by addition of nontemplated uridines if the mRNA is further trimmed by 3′hExo. These mRNAs are tightly cell-cycle regulated, and a critical regulatory step is rapid degradation of the histone mRNAs when DNA replication is inhibited. An initial step in histone mRNA degradation is digestion 2–4 nt into the stem by 3′hExo and uridylation of this intermediate. The mRNA is then subsequently degraded by the exosome, with stalled intermediates being uridylated. The enzyme(s) responsible for oligouridylation of histone mRNAs have not been definitively identified. Using high-throughput sequencing of histone mRNAs and degradation intermediates, we find that knockdown of TUT7 reduces both the uridylation at the 3′ end as well as uridylation of the major degradation intermediate in the stem. In contrast, knockdown of TUT4 did not alter the uridylation pattern at the 3′ end and had a small effect on uridylation in the stem–loop during histone mRNA degradation. Knockdown of 3′hExo also altered the uridylation of histone mRNAs, suggesting that TUT7 and 3′hExo function together in trimming and uridylating histone mRNAs

Formation of the 3′ end of histone mRNA: Getting closer to the end

Nearly all eukaryotic mRNAs end with a poly (A) tail that is added to their 3’ end by the ubiquitous cleavage/polyadenylation machinery. The only known exception to this rule are metazoan replication dependent histone mRNAs, which end with a highly conserved stem-loop structure. This distinct 3’ end is generated by specialized 3’end processing machinery that cleaves histone pre-mRNAs 4–5 nucleotides downstream of the stem-loop and consists of the U7 small nuclear RNP (snRNP) and number of protein factors. Recently, the U7 snRNP has been shown to contain a unique Sm core that differs from that of the spliceosomal snRNPs, and an essential heat labile processing factor has been identified as symplekin. In addition, cross-linking studies have pinpointed CPSF-73 as the endonuclease, which catalyzes the cleavage reaction. Thus, many of the critical components of the 3’ end processing machinery are now identified. Strikingly, this machinery is not as unique as initially thought but contains a number of factors involved in cleavage/polyadenylation, suggesting that the two mechanisms have a common evolutionary origin. The greatest challenge that lies ahead is to determine how all these factors interact with each other to form a catalytically competent processing complex capable of cleaving histone pre-mRNAs

Novel localization and possible functions of cyclin E in early sea urchin development

In somatic cells, cyclin E-cdk2 activity oscillates during the cell cycle and is required for the regulation of the G1/S transition. Cyclin E and its associated kinase activity remain constant throughout early sea urchin embryogenesis, consistent with reports from studies using several other embryonic systems. Here we have expanded these studies and show that cyclin E rapidly and selectively enters the sperm head after fertilization and remains concentrated in the male pronucleus until pronuclear fusion, at which time it disperses throughout the zygotic nucleus. We also show that cyclin E is not concentrated at the centrosomes but is associated with condensed chromosomes throughout mitosis for at least the first four cell cycles. Isolated mitotic spindles are enriched for cyclin E and cdk2, which are localized to the chromosomes. The chromosomal cyclin E is associated with active kinase during mitosis. We propose that cyclin E may play a role in the remodeling of the sperm head and re-licensing of the paternal genome after fertilization. Furthermore, cyclin E does not need to be degraded or dissociated from the chromosomes during mitosis; instead, it may be required on chromosomes during mitosis to immediately initiate the next round of DNA replication

The TATA Binding Protein in the Sea Urchin Embryo Is Maternally Derived

AbstractThe cDNA encoding the TATA binding protein was isolated from 8- to 16-cell and morula-stage embryonic libraries of two distantly related species of sea urchin,Strongylocentrotus purpuratusandLytechinus variegatus,respectively. The two proteins are 96% identical over both the N- and C-terminal domains, suggesting a conservation of transcriptional processes between the two species. The prevalence of SpTBP transcripts at several developmental time points was determined using the tracer excess titration method, and the corresponding number of TBP protein molecules was determined by quantitative Western blot analysis. Our results indicate that the amount of TBP mRNA and protein per embryo remains relatively constant throughout development. An initial large pool of TBP protein (>109) molecules in the egg becomes diluted as a consequence of cell division and decreases to about 2 × 106molecules per cell by the gastrula stage. We found byin situRNA hybridization that the oocyte contains a large amount of TBP mRNA which is depleted late in oogenesis so that the eggs and early embryos have extremely low levels of TBP mRNA. We conclude that the oocyte manufactures nearly all of the TBP protein necessary for embryogenesis

Cyclin E and Its Associated cdk Activity Do Not Cycle during Early Embryogenesis of the Sea Urchin

Female sea urchins store their gametes as haploid eggs. The zygote enters S-phase 1 h after fertilization, initiating a series of cell cycles that lack gap phases. We have cloned cyclin E from the sea urchin Cyclin E is synthesized during oogenesis, is present in the germinal vesicle, and is released into the egg cytoplasm at oocyte maturation. Cyclin E synthesis is activated at fertilization, although there is no increase in cyclin E protein levels due to continuous turnover of the protein. Cyclin E protein levels decline in morula embryos, while cyclin E mRNA levels remain high. After the blastula stage, cyclin E mRNA and protein levels are very low, and cyclin E expression is predominant only in cells that are actively dividing. These include cells in the left coelomic pouch, which forms the adult rudiment in the embryo. The cyclin E present in the egg is complexed with a protein kinase. Activity of the cyclin E/cdk2 changes little during the initial cell cycles. In particular, cyclin E-cdk2 levels remain high during both S-phase and mitosis. Our results suggest that progression through the early embryonic cell cycles in the sea urchin does not require fluctuations in cyclin E kinase activity