Masami Nozaki

In Vitro and In Vivo Gene Delivery by Recombinant Baculoviruses

ABSTRACT Although recombinant baculovirus vectors can be an efficient tool for gene transfer into mammalian cells in vitro, gene transduction in vivo has been hampered by the inactivation of baculoviruses by serum complement. Recombinant baculoviruses possessing excess envelope protein gp64 or other viral envelope proteins on the virion surface deliver foreign genes into a variety of mammalian cell lines more efficiently than the unmodified baculovirus. In this study, we examined the efficiency of gene transfer both in vitro and in vivo by recombinant baculoviruses possessing envelope proteins derived from either vesicular stomatitis virus (VSVG) or rabies virus. These recombinant viruses efficiently transferred reporter genes into neural cell lines, primary rat neural cells, and primary mouse osteal cells in vitro. The VSVG-modified baculovirus exhibited greater resistance to inactivation by animal sera than the unmodified baculovirus. A synthetic inhibitor of the complement activation pathway circumvented the serum inactivation of the unmodified baculovirus. Furthermore, the VSVG-modified baculovirus could transduce a reporter gene into the cerebral cortex and testis of mice by direct inoculation in vivo. These results suggest the possible use of the recombinant baculovirus vectors in combination with the administration of complement inhibitors for in vivo gene therapy.

Epigenetic Regulation of Mouse Sex Determination by the Histone Demethylase Jmjd1a

More Determined Sex Although several transcription factors participate in mammalian sex determination, the contribution from specific epigenetic regulation is just being revealed. Kuroki et al. (p. 1106) show that a JmjC domain–containing protein, Jmjd1a, catalyzes H3K9 demethylation of the Y-linked sex-determining gene Sry in mice to enable its expression above the required threshold level. Ablation of Jmjd1a function results in mouse male-to-female sex reversal, hence not only revealing a mechanism of Sry regulation but also the pivotal role of epigenetic regulation in mammalian sex determination. Histone modification controls mammalian sex determination. Developmental gene expression is defined through cross-talk between the function of transcription factors and epigenetic status, including histone modification. Although several transcription factors play crucial roles in mammalian sex determination, how epigenetic regulation contributes to this process remains unknown. We observed male-to-female sex reversal in mice lacking the H3K9 demethylase Jmjd1a and found that Jmjd1a regulates expression of the mammalian Y chromosome sex-determining gene Sry. Jmjd1a directly and positively controls Sry expression by regulating H3K9me2 marks. These studies reveal a pivotal role of histone demethylation in mammalian sex determination.

Isolation and characterization of cDNA clones specifically expressed in testicular germ cells.

We have cloned cDNAs involved in germ cell‐specific expression. For this, a subtracted cDNA library was generated by subtracting cDNAs derived from supporting cells of mutant testis from wild‐type testis cDNAs. Detailed analyses of mRNA expression revealed that the genes corresponding to the cloned cDNAs were exclusively expressed in testes and were developmentally controlled.

Identification and characterization of testis specific ornithine decarboxylase antizyme (OAZ-t) gene: expression in haploid germ cells and polyamine-induced frameshifting

Polyamines are known to play important roles in the proliferation and differentiation of many types of cells. However, in the testis, where polyamines such as spermidine and spermine exist in high concentrations, their roles still remains to be elucidated.

Cloning and sequencing of the gene coding for dextranase from Streptococcus salivarius.

We cloned and sequenced the dextranase (Dex) (1,6-alpha-glucanhydrolase; EC 3.2.1.11)-encoding gene from Streptococcus salivarius (Ss) strain M-33. Recombinant clones from an Ss genomic library specifying Dex activity were identified as colonies surrounded by transparent halos on blue dextran plates. One of the clones had a 4.3-kb KpnI fragment containing the gene coding for an 826-amino-acid polypeptide with a molecular mass of 87.9 kDa, which corresponds well to that of native Dex from the Ss culture supernatant. There was no sequence homology between the gene encoding Ss Dex and the gene encoding dextran glucosidase of S. mutans, or between their protein products.

Isolation and Characterization of a Haploid Germ Cell-Specific Novel Complementary Deoxyribonucleic Acid; Testis-Specific Homologue of Succinyl CoA:3-Oxo Acid CoA Transferase

Abstract We have isolated a cDNA clone encoding a mouse haploid germ cell-specific protein from a subtracted cDNA library. Sequence analysis of the cDNA revealed high homology with pig and human heart succinyl CoA:3-oxo acid CoA transferase (EC 2.8.3.5), which is a key enzyme for energy metabolism of ketone bodies. The deduced protein consists of 520 amino acid residues, including glutamate 344, known to be the catalytic residue in the active site of pig heart CoA transferase and the expected mitochondrial targeting sequence enriched with Arg, Leu, and Ser in the N-terminal region. Thus, we termed this gene scot-t (testis-specific succinyl CoA:3-oxo acid CoA transferase). Northern blot analysis, in situ hybridization, and Western blot analysis demonstrated a unique expression pattern of the mRNA with rapid translation exclusively in late spermatids. The scot-t protein was detected first in elongated spermatids at step 8 or 9 as faint signals and gradually accumulated during spermiogenesis. It was also detected in the midpiece of spermatozoa by immunohistochemistry. The results suggest that the scot-t protein plays important roles in the energy metabolism of spermatozoa.

Transient expression analysis of the mouse ornithine decarboxylase antizyme haploid-specific promoter using in vivo electroporation

The testicular isoform of the ornithine decarboxylase antizyme (OAZt) gene is expressed exclusively in the haploid spermatids of mice. The 357‐bp region, which includes a TATA‐less promoter and an untranslated region, is sufficient for OAZt gene expression in the spermatids of transgenic mice. In this study, in vivo transient transfection to living mouse testes was used to define the transcriptional regulatory elements of the OAZt gene promoter. We found that the 10‐bp element that contains an initiator (Inr) plays a central role as the core promoter, in combination with a downstream element, while two cyclic adenosine monophosphate‐responsive element (CRE)‐like sites in the upstream region also contribute to promoter activity. The electrophoretic mobility shift assay showed binding of the testis‐specific factors to these elements. Our results show that the in vivo DNA transfer technique enables detailed analysis of haploid germ cell‐specific gene regulation in mice.

Molecular Cloning and Characterization of Phosphatidylcholine Transfer Protein-Like Protein Gene Expressed in Murine Haploid Germ Cells

Abstract We have isolated a cDNA clone specifically expressed in spermiogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1392 nucleotides and had an open reading frame of 873 nucleotides encoding a protein of 291 amino acid residues. Computer-mediated homology search revealed that the nucleotide sequence was unique but the deduced amino acid sequence had similarity to mouse phosphatidylcholine transfer protein (PCTP). We named this newly isolated gene PCTP-like protein. Northern blot analysis revealed a 1.4-kilobase mRNA expressed in the testis, kidney, liver, and intestine with the highest level in the testis. Messenger RNA expression in the testis was detected first on Day 23 in postnatal development and then increased up to adulthood. The protein, having a molecular weight of approximately 40 000, was encoded by the mRNA and was detected at the tail of the elongated spermatids and sperm by immunohistochemical staining.

Gene expression of cytokeratin endo A and endo B during embryogenesis and in adult tissues of mouse

We have examined the pattern of gene expression of mouse cytokeratin endo A and endo B during postimplantational development and in adult organs by Northern blot and in situ hybridization analyses. Both mRNAs localized in the ectoplacental cone, trophoblastic giant cells surrounding the parietal yolk sac, trophoblast cells in placenta, visceral yolk sac, and simple epithelium of the embryo during postimplantational development and in simple or transitional epithelial tissues in adult organs. These results indicate that endo A and endo B are coexpressed and may play some roles in these tissues.

Activation of the mouse cytokeratin A (endo A) gene in teratocarcinoma F9 cells by the histone deacetylase inhibitor Trichostatin A

Treatment of cultured cells with sodium butyrate, that is the histone deacetylase inhibitor, induces the histone hyperacetylation and the expressions of various mammalian genes without affecting the level of protein synthesis. However, butyrate is a non‐specific inhibitor of deacetylase because of its effects on various other enzymes and nuclear proteins other than histones. On the other hand, Trichostatin A (TSA) was recently found to be a potent and specific inhibitor of histone deacetylase. We examined the effect of TSA on the expression of mouse cytokeratin A (endo A). TSA increased endo A expression in F9 cells, and was efffective at a much lower concentration than sodium butyrate. We also examined the changes of chromatin structure induced by the two drugs by a DNase I‐hypersensitivity assay. Both drugs induced the formation of a DNase I‐hypersensitive site (DH site) in only the promoter region. The precise mechanism(s) by which the two drugs increase endo A gene expression is unknown, but these results suggest that endo A expression is induced by inhibition of histone deacetylase and that the effect is at the transcriptional level.