小鼠精子注入兔卵母细胞受精研究

The methods of intracytoplasmic sperm injection (ICSI) and subzonal injection (SUZI) were used to study heterologous fertilization and embryonic development between the mouse and the rabbit. Results were as follows: 1. The mouse sperm nuclei decondensed and formed pronuclei following microinjection into cytoplasm and perivitelline space (PVS) of rabbit oocytes; 2. The hybrid embryos developed to the stage of 8-cell when cultured in vitro; 3. The karyotype analysis showed a normal complement of rabbit oocyte and mouse sperm chromosomes in the 4-cell hybrid embryos; 4. The ultrastructure of 4-cell hybrid embryos was similar to that of normal 4-cell rabbit embryos; 5. The fertilization rate (32.4%) and cleavage rate (22.2%) when 5-10 mouse spermatozoa were injected were higher than those of injection of a single spermatozoon into PVS of the rabbit oocyte, but the difference was not significant (P > 0.05). The fertilization rate (42.3%) and cleavage rate (30.8%) in rabbit oocytes in vitro matured for 11-12 h were higher than those in the oocytes which were in vitro matured for 24-25 h following microinjection of 1-2 mouse spermatozoa into PVS, but the difference was not significant (P > 0.05).     

Periodicity in the length of 3'-poly(A) tails from native globin mRNA of rabbit.

Globin mRNA from rabbit reticulocytes was labelled at the 3'-end with [5'-32P]pCp by T4 RNA ligase. The 3'-poly(A) tail was released by digestion of mRNA with T1 ribonuclease and its size distribution determined by gel electrophoresis and autoradiography. The length of the 3'-poly(A) tails varied from about 15-150 residues, but the size distribution exhibited peaks in the abundance of poly(A) species at intervals of approx. 25 residues. This periodicity appears to reflect the manner in which proteins bind to the 3'-poly(A) tail. The function of such regular interactions may be to control mRNA breakdown in the cytoplasm.     

Requirement for 7-methylguanosine in translation of globin mRNA in vivo.

The 7-methylguanosine (m7G) residue present in the m7G5' ppp5'X-"CAP" structure of rabbit globin mRNA was removed quantitatively by periodate oxidation followed by beta-elimination in the presence of cyclohexylamine. The RNA thus treated was intact and exhibited no signs of degradation as examined by polyacrylamide gel electrophoresis in formamide. Assay for protein synthesis using a wheat germ cell-free system showed that the globin mRNA lacking m7G had lost most of its messenger activity. Identical treatment, of satellite tobacco necrosis virus (STNV) RNA, which does not contain the 5'-terminal "CAP" structure, resulted in no loss of its mRNA activity. Since the importance of the m7G residue in eukaryotic mRNA has not yet been shown essential for translation in vivo, both untreated and treated globin mRNAs were injected into frog oocytes and their translation into globin was measured at intervals over a ninety-six hour period. Globin mRNA either treated with periodate alone or lacking in m7g altogether were both found to have lost more than 90% of their activity in vivo.     

Analysis of adenovirus early region 4-encoded polypeptides synthesized in productively infected cells.

Peptide-specific antisera were developed to analyze the products encoded by adenovirus type 5 early region 4 (E4) open reading frames 6 and 7. Reading frame 6 previously was shown to encode a 34-kilodalton polypeptide (34K polypeptide) that forms a complex with the early region 1B (E1B)-55K antigen and is required for efficient viral growth in lytic infection. Antisera that were generated recognized the E4-34K protein as well as a family of related polypeptides generated by the fusion of open reading frames 6 and 7. These polypeptides shared amino-terminal sequences with the 34K protein. Short-pulse analysis suggested that the heterogeneity observed with the 6/7 fusion products resulted from differential splicing patterns of related E4 mRNAs. An antiserum directed against the amino terminus of reading frame 6 recognized only the free form of the 34K antigen that was not associated with the E1B-55K protein. This observation allowed the determination of the stability of the free and complexed form of this polypeptide. Pulse-chase analyses demonstrated that both forms of the 34K protein had half-lives greater than 24 h, suggesting that complex formation did not result in stabilization of this gene product. The half-lives of the 6/7 fusion products were approximately 4 h. The 34K protein also was shown to have a nuclear localization within infected cells. Finally, analysis of a mutant carrying deletions in both the E4-34K and E1B-55K polypeptides indicated that the complex formed between these two proteins was a functional unit in lytic infection.     

Increase in globin chains and globin mRNA in erythroleukemia cells in response to hemin.

Addition of 50 μm hemin to mouse erythroleukemia cells cultured in 0.5% dimethyl-sulfoxide (DMSO) resulted in >10-fold stimulation of globin chain synthesis as a percentage of acid precipitable protein. In cultures fully induced with 1.5% DMSO, addition of 15 mm 3-amino-1,2,4-triazole (AT), an inhibitor of heme synthesis, reduced globin chain synthesis to uninduced levels and reduced globin mRNA levels to less than 20% of induced values. The inhibition of AT was prevented by simultaneous addition of 25 μm hemin to the cultures. Using RNA-DNA hybridization analysis, the amount of globin mRNA sequences as a fraction of total cytoplasmic RNA was also increased by addition of 50 μm hemin to cultures with 0.5% DMSO. The results suggest that exogenous hemin can promote globin chain synthesis, that endogenously synthesized heme can be required for globin chain synthesis, and that hemin directly or indirectly also alters the appearance or degradation of globin mRNA sequences in the cytoplasm.     

Comparative inhibition of rabbit globin mRNA translation by modified antisense oligodeoxynucleotides.

We have studied the translation of rabbit globin mRNA in cell free systems (reticulocyte lysate and wheat germ extract) and in microinjected Xenopus oocytes in the presence of anti-sense oligodeoxynucleotides. Results obtained with the unmodified all-oxygen compounds were compared with those obtained when phosphorothioate or alpha-DNA was used. In the wheat germ system a 17-mer sequence targeted to the coding region of beta-globin mRNA was specifically inhibitory when either the unmodified phosphodiester oligonucleotide or its phosphorothioate analogue were used. In contrast no effect was observed with the alpha-oligomer. These results were ascribed to the fact that phosphorothioate oligomers elicit an RNase-H activity comparable to the all-oxygen congeners, while alpha-DNA/mRNA hybrids were a poor substrate. Microinjected Xenopus oocytes followed a similar pattern. The phosphorothioate oligomer was more efficient to prevent translation than the unmodified 17-mer. Inhibition of beta-globin synthesis was observed in the nanomolar concentration range. This result can be ascribed to the nuclease resistance of phosphorothioates as compared to natural phosphodiester linkages, alpha-oligomers were devoid of any inhibitory effect up to 30 microM. Phosphorothioate oligodeoxyribonucleotides were shown to be non-specific inhibitors of protein translation, at concentrations in the micromolar range, in both cell-free systems and oocytes. Non-specific inhibition of translation was dependent on the length of the phosphorothioate oligomer. These non-specific effects were not observed with the unmodified or the alpha-oligonucleotides.     

Identification of globin mRNA in 10s RNA of rabbit reticulocytes

Electrophoresis on 6% polyacrylamide gels splits 10s RNA of detergenttreated polysomes from rabbit reticulocytes into two major bands. After these two RNAs are isolated separately, the first 10s RNA¹ directs the synthesis of both α and β chains in the Krebs II ascites cell-free system. In contrast, the second 10s RNA is inactive in directing globin synthesis. This result is further documented by separation of the two 10s RNAs by oligo dT-cellulose chromatography and by isolation of globin mRNA after EDTA-treatment of reticulocyte polysomes. Therefore, globin mRNA containing both α- and β-chain synthetic capacity moves as a single RNA species on electrophoresis in polyacrylamide gels.     

In vivo incorporation of different amino acids into electrophoretically characteristic polypeptides synthesized by HeLa cell mitochondria

The $\text{in vivo}$ incorporation of each of the twenty common amino acids into electrophoretically characteristic polypeptides synthesized by HeLa cell mitochondria has been investigated. Under labeling conditions which allow translation only on mitochondrial ribosomes, incorporation of all the amino acids, except aspartic acid, cysteine, glutamic acid and glycine, has been detected. These exceptions are probably due to problems related to amino acid pool size and/or equilibration.     

Stability of rabbit globin and its messenger RNA in the mouse ovum

Fertilized one-cell mouse ova were injected with rabbit globin messenger RNA, (mRNA) containing approximately equal quantities of α- and β-message. The half-lives of α- and β-globin were 8.2 and 7.0 h respectively. The half-lives of α- and β-globin messages were 8.8 and 9.5 h respectively. Approx. four times as much α- as β-globin was present in the ova following the injections.     

Red cell ghost-mediated microinjection of RNA into HeLa cells: I. A comparison of two techniques for the entrapment and microinjection of tRNA and mRNA

We have compared two techniques for introducing RNA into red blood cell ghosts. In the pre-swell technique, RNA is introduced into red cells without prior removal of endogenous contents. In the multiple lysis technique, the red cells are subjected to two or three cycles of reversible lysis, prior to introducing the RNA, in order to first remove the normal red cell constituents. The pre-swell technique offers much greater entrapment of both tRNA and protamine messenger RNA (mRNA), but the RNA appears to be degraded during the procedure. This may be due either to nuoleolytic degradation or oxidation by the high concentration of endogenous hemoglobin. The multiple lysis technique offers much lower entrapment but also results in diminished degradation of the entrapped RNA. Although some degradation is apparent, a significant portion of the biological activity of the entrapped protamine mRNA is retained. We have also fused red cells loaded with protamine mRNA by the multiple lysis technique to HeLa cells using polyethylene glycol 6000. The recipient HeLa cells are capable of translating this heterologous message into protamine, a trout testis chromosomal protein.     

The separation of 9 S RNA from avian immature red blood cells into f2c-histone mRNA and globin mRNA.

9 S RNA from avian immature red blood cells was isolated from polysome-released ribonucleoprotein particles by sucrose-gradient techniques. Translation of this RNA in an Ehrlich ascites cell-free system and product analysis revealed that globin mRNA was contaminated by f2c-histone mRNA. When 9 S RNA was applied to oligo(dT)-cellulose columns a partial separation could be achieved. Poly (A)-containing globin mRNA did not contain f2c-histon mRNA, whereas the RNA which was not absorbed to oligo(dT)-cellulose contained all the f2c-histone mRNA besides substantial amounts of globin mRNA.     

Position specific growth regulation of 3T3 cells in vivo.

In this study we have investigated the mechanism by which spatial growth is regulated by monitoring 3T3 cells, introduced into the developing mouse limb using exo utero surgery. The 3T3 cells were labeled with a human cell surface glycoprotein, CD8, and injected into stage 7-9 mouse limbs. At 24 and 48 hr after injection embryos were labeled with [3H]thymidine and processed for immunohistochemistry and autoradiography. The labeling index of CD8 positive cells was compared to that of neighboring limb bud cells and also to the position of the injection site within the limb. We find that the labeling index of 3T3 cells is in accord with that of the limb cells that immediately surround them; 3T3 cells display a high labeling index in limb regions of high growth and a low labeling index in limb regions of low growth. In addition, we find that both limb bud cells and injected 3T3 cells display a general proximal (low) to distal (high) gradient of growth at the stages analyzed. We conclude from these results that position-specific regulation of growth occurs in a non-cell autonomous manner and is likely to be mediated by mitogenic signals that are localized within the limb environment. In addition, our results demonstrate the usefulness of utilizing established cell lines as in vivo probes to monitor developmental mechanisms.     

An inhibitor(s) of globin mRNA translation in rabbit serum

1. A factor found in rabbit serum inhibits globin mRNA translation in vitro. 2. Inhibition of globin mRNA translation has been demonstrated in a cell-free rabbit reticulocyte lysate. 3. The inactivation of globin mRNA translation is not attributed to either serum albumin or ribonuclease activities. 4. Dialyzing the inhibitor for 24 hr at 4 degrees C does not result in the diminution of the inhibiting activity. However, the activity of the inhibitor is destroyed by heating to 70-80 degrees C for 5 min or by treatment with trypsin for 2 hr. 5. Ion exchange chromatography points to the inhibitor being a neutral protein, whereas, polyacrylamide gel electrophoresis reveals one major band with mol. wt 43 kDa. 6. The activity of the inhibiting material 3-fold greater in anemic serum than in normal serum. 7. These studies suggest that rabbit serum contains a protein inhibitor that may play a physiological role in regulating protein synthesis in red cells.     

Transfer of human globin genes to erythroleukemic mouse cells.

Thymidine kinase negative (TK-) Friend cells were transformed with recombinant molecules carrying human globin genes and the thymidine kinase gene of herpes simplex virus type 1 DNA. Transformation frequencies of 1 transformant/microgram donor DNA/1 x 10(6) cells were obtained by standard procedures and this was increased 20- to 30-fold by treating recipient cells with dimethyl sulfoxide or glycerol. Transformed cell lines expressed thymidine kinase activity of viral origin as determined by its insensitivity to 0.2 mM dTTP and electrophoretic mobility in polyacrylamide gels. The physical status of donor DNA in the transformed cells was examined in Hirt precipitates and supernatants by Southern blot hybridization and spot hybridization techniques. This analysis showed that most donor sequences were present in a circular or concatenate configuration, but also was suggestive of some donor sequences being integrated into high molecular weight DNA. Expression of human globin genes and particularly the epsilon-globin gene in the transformed Friend cells was studied by Northern blot hybridization analysis.     

Evidence that the 5' end of rabbit globin mRNA is hybridized with its 3' end

• 1.1. A radiopolyadenylated rabbit globin mRNA was treated with different concentrations of ribonuclease V₁ from cobra venom.
• 2.2. The enzymatic digests were chromatographed on an aminophenylboronate-agarose column, which specifically captured the cap structure i.e. n⁷G(5') ppp (5') NmP.
• 3.3. When the capture fragment was chromatographed on a Sephadex G-100 column, its size was smaller than the native molecule and also bore radioactivity, i.e. a poly(A) tail.
• 4.4. These results provide evidence that the 5' end (which encompasses the cap structure) of rabbit globin mRNA is hybridized and in close proximity to its 3' end.
• 5.5. We conclude that this conformation is required for messenger translation efficiency.