Isolation and transformation of primary mesenchymal cells of the chick embryo.

Pure primary mesenchymal cells from definitive streak stage chick embryos have been prepared free of epiblast and hypoblast cells. These cells have the potential in culture to differentiate into erythroid cells, beating heart muscle tissue, chondrocytes and epithelial cells. Transformation in vitro of pure primary mesenchymal cells by avian erythroblastosis virus (wt-AEV) and a temperature-sensitive mutant (ts34-AEV) gave rise to rapidly growing cells which remained largely undifferentiated, could be cloned in semi-solid medium and could be maintained for up to 3 months in culture. The majority of mesenchymal cells transformed by wt-AEV (MAE cells) are benzidine-negative. Gel electrophoresis of radioactively labeled cell proteins, immunoprecipitated with specific antisera against chicken hemoglobin, showed that MAE cell clones synthesize the alpha D, pi (or pi') and some unidentified "globin" polypeptide chains. Treatment of MAE cell clones with 1.0 mM n-butyrate stops cell proliferation reversibly and causes an increased synthesis of alpha D and pi (or pi') globin polypeptide chains. In certain clones of mesenchymal cells transformed by a temperature-sensitive mutant of the virus, ts34-AEV (MAE-ts34 cells), benzidine-positive cells can be induced by a shift from 37 degrees to 41 degrees C. The ability of the clone to undergo an increase in benzidine-positivity by temperature shift is decreased with the age of the clone. Different clones show a variable proportion of cells which are positive by immunofluorescence for both globin and chicken-specific histone H5. The alpha A and alpha D globin chains are synthesized in MAE-ts34 clones, but the ratios and quantities of these chains vary for different clones. Temperature shift made little difference in the types and quantities of globin chains synthesized; the increase in benzidine positivity is probably due to an increase in heme biosynthesis.     

Hormone-dependent terminal differentiation in vitro of chicken erythroleukemia cells transformed by ts mutants of avian erythroblastosis virus

Chicken erythroblast cell strains and a cell line transformed by ts mutants of avian erythroblastosis virus (AEV) terminally differentiate when shifted to the nonpermissive temperature (42°C). The differentiated cells resemble mature erythrocytes with respect to morphology and ultrastructure, expression of differentiation-specific cell-surface antigens, pattern of protein synthesis and hemoglobin content. Terminal differentiation is dependent on conditions favoring the differentiation of normal erythroid progenitor cells, including an erythropoietin-like factor. Colonies of ts AEV cells grown at 42°C in semisolid medium resemble erythrocyte colonies derived from normal erythroid progenitor cells. The colonies obtained were comparable in size or slightly larger than the late erythroid precursor (CFU-E) colonies. These results suggest that AEV-transformed cells are blocked at a stage of differentiation that is more advanced than that of the uninfected target cells. ts AEV cells are irreversibly committed to terminal differentiation within 20 to 30 hr after shift to 42°C.     

Expression of Embryonic Haemoglobin in ts AEV-Transformed Embryonic Erythroid Cells During Temperature-Induced Differentiation

Cells prepared from 1-day-old chick blastoderms were infected with a temperature-sensitive mutant of avian erythroblastosis virus ( ts AEV). Clonal strains of transformed erythroblasts were isolated from the infected blastoderm cells. By shift to the nonpermissive temperature, these cells could be induced to differentiate into erythrocyte-like cells which expressed embryonic haemoglobins. Embryonic haemoglobins could not be detected in ts AEV-transformed erythroblasts from adult bone marrow when induced to differentiate under the same conditions. In contrast to normal primitive erythrocytes, ts AEV-infected embryonic erythroblasts differentiated in vitro expressed also adult haemoglobin. These results suggest an influence of the haematopoietic environment on the switch from embryonic to adult erythrocytes.     

Kinetics of ligand binding to ferrous heme groups of hemoglobin MSaskatoon.

Hemoglobin M_Saskatoon (α₂^Aβ₂^63tyr) has two α chains in the normal ferrous state, while its two β chains are in the ferric state. The reaction of hemoglobin M_Saskatoon with carbon monoxide at pH 7 and 20 °C in the presence and absence of dithionite was studied. In the absence of dithionite only the α chains react and the combination rate is slow and similar to that of normal deoxyhemoglobin. After the addition of dithionite the rate of reaction is greatly increased initially and then decreases to a rate similar to that seen in the absence of dithionite. The dissociation of oxygen from hemoglobin M_Saskatoon at pH 7 and 20 °C was found for the α subunits to be similar to that seen for normal oxyhemoglobin. This similarity in the kinetic properties of normal hemoglobin and the α subunits of hemoglobin M_Saskatoon in both ligand combination and dissociation reactions indicates that the α subunits of hemoglobin M_Saskatoon undergo a structural transition from a low to high affinity form on liganding. Since the β subunits react rapidly with carbon monoxide even when the α subunits are unliganded, it appears that the ligand binding sites of the β chains are uncoupled from the state of liganding of the α subunits.     

The translation of rabbit hemoglobin messenger RNA in a cell-free extract from chick embryo brain

The translation of rabbit hemoglobin messenger RNA in an unfractionated cytoplasmic extract from chick embryo brain was studied. This translation was not dependent upon reticulocyte-specific factors. An analysis of the product synthesized in vitro with the embryo brain cell-free extract and rabbit hemoglobin messenger RNA by carboxymethyl cellulose chromatography showed that the system was capable of synthesizing both the α and β globin chains. Analysis of the tryptic peptides of the in vitro synthesized α chain by ion-exchange chromatography showed that the embryo brain extract with rabbit hemoglobin messenger RNA was capable of synthesizing the complete α chain of rabbit hemoglobin. The results suggest that no stringent tissue-specific controls exist for the translation of globin messenger RNA and were discussed in this context.     

Phosphorylation of talin at tyrosine in Rous sarcoma virus-transformed cells.

The cytoskeletal protein talin was found to undergo enhanced phosphorylation at tyrosine residues in chicken embryo fibroblasts following transformation by Rous sarcoma virus. An increase in the tyrosine phosphorylation of talin was also observed within 6 h in cells infected by the temperature-sensitive mutant tsNY68 after a shift from the nonpermissive to the permissive temperature. The overall extent of phosphorylation was 0.07 mol of phosphate per mol of talin and was not appreciably altered by transformation. In uninfected cells talin was shown to be phosphorylated at multiple sites by tryptic peptide mapping. Following transformation most of these sites remained phosphorylated, to the same or to a lesser extent, while novel, phosphotyrosine-containing phosphopeptides appeared. Talin was phosphorylated at tyrosine in cells infected by Rous sarcoma virus mutants which induce altered or partial transformation morphologies; thus the increased phosphorylation of talin at tyrosine occurred irrespective of the morphology induced. Transformation by Y73 also induced elevated levels of phosphotyrosine in talin, whereas transformation by the avian erythroblastosis and Fujinami sarcoma viruses did not.     

Determination of the primary sequence of the duck αD globin mRNA and comparison of all adult duck and chick globin mRNA sequences

The nucleotide sequence of the duck α^D globin mRNA was determined. Its main feature is an exceptionally short 3′ non-coding segment of only 46 nucleotides, placed after the coding sequence of 141 codons. The last of the 6 adult globin mRNA of duck and chicken being thus sequenced, a comparison of all their features has become possible. Comparing the duck α^D mRNA to the related sequence in the chicken, we found greater homology than comparing it to the linked α^A globin sequence in the same species. Extensive homology can be found for a same globin chain α^A, α^D or β in between different avian species including also the goose and the ostrich; the avian α globin chains show a lower degree of sequence conservation in between species than the β chains. In contrast, within one species the three globin sequences have further diverged. The divergence between the α^A and α^D globin within a same species point to individual functional specificity and hence independent evolution and suggest that a mechanism of ‘gene conversion’ did not operate in between the avian α globin genes. Two segments of the amino acid sequence which we named ‘A^α’ and ‘B^α’ remain homologous in all avian α globins; two other regions ‘A^β’ and ‘B^β’ are identical in between the β globins. Segment A is placed at the 5′ end of exon II, and segment B at the 3′ end of the same exon; some amino acids in those segments are involved in the Heme binding site. Being almost identical in all know mammalian and avian globins of the α respectively the β type, regions A and B seem to represent the best conserved sequences in adult globin mRNA maintained during the divergence of species.     

Temperature-dependent transmembrane potential changes in cells infected with a temperature-sensitive moloney sarcoma virus

Normal rat kidney cells (NRK) infected with the temperature-sensitive (ts) transformation mutant of Moloney murine sarcoma virus yielded a clone of cells, 6m2, that exhibited a transformed morphology at 33°C and a normal morphology at 39°C. Transmembrane potential (Em) was measured fluorometrically using a cyanine dye diS-C₃-(5). Fluorescence was inversely correlated with Em. Cells at 33°C had lower Em. Em changes were recorded within 15 minutes of temperature shift from 33°C to 39°C in both directions, increasing in the 33°C to 39°C direction and decreasing in the 39°C to 33°C direction. Uninfected NRK cells when shifted under the same condition exhibited small fluorescence changes in the 33°C to 39°C direction. Shifting from 39°C to 33°C resulted in Em changes similar to those in 6m2 cells. Also studied was a cell line infected with a spontaneous revertant of the ts mutant, designated 54-5A4; it was transformed at both temperatures. Shifting from 33°C to 39°C in both directions yielded small changes. Transmembrane potential changes in 6m2 cells precede other transformation-specific changes that occur after a temperature shift.     

Temperature-sensitive mutants of avian erythroblastosis virus: surface expression of the erbB product correlates with transformation

The v-erbB gene of avian erythroblastosis virus (AEV) codes for an integral plasma membrane glycoprotein, gp74erbB. Expression of gp74erbB and its intracellular precursors, gp66erbB and gp68erbB, has been studied in cells transformed by two temperature-sensitive mutants of AEV. After shift to 42 degrees C, the processing of gp68erbB is blocked in tsAEV-transformed, but not in wtAEV-transformed, erythroblasts and fibroblasts. In addition, gp74erbB disappears from the surface of tsAEV cells within 12 hr after shift. Thus tsAEV mutants probably bear a lesion in v-erbB that affects the maturation and subcellular localization of gp74erbB. The tsAEV erythroblasts, when "committed" to differentiation by a pulse-shift to 42 degrees C, reexpress gp74erbB during terminal differentiation at 36 degrees C. This suggests that tsAEV erythroblasts become insensitive to the transforming functions of gp74erbB at a certain stage of differentiation.     

Separation of alpha and beta chains of rabbit globin using SDS-polyacrylamide gel electrophoresis

A procedure to separate the α and β globin chains of rabbit hemoglobin, denatured with sodium dodecyl sulfate in the presence of mercaptoethanol, on a column of polyacrylamide gel was developed. The identity of the two separated chains was verified by (a) differences in distribution of radioactivity between the chains when the hemoglobin samples were labeled uniformly with various ³H- or ¹⁴C-labeled amino acids; (b) the analysis of the chain distribution of radioactivity in purified hemoglobin isolated from rabbit reticulocytes, pulse-labeled with [³H] leucine; and (c) the separation pattern of a mixture of authentic [α-³H]- and [β-¹⁴C]-labeled globin chains. The globin chains of human hemoglobin A also could be separated in a similar manner. This procedure is particularly useful when only microgram quantities of hemoglobin are available for study.     

Competition between α and β globin messenger RNA

Addition to an unfractionated reticulocyte lysate of either α or β globin mRNA or reticulocyte initiation factors does not alter the overall rate of globin synthesis. Addition of β mRNA results in enhanced synthesis of β product and decreased production of α; conversely, addition of α mRNA results in enhanced synthesis of α globin and decreased production of β. We conclude that the amount of any putative α mRNA or β mRNA-specific factor does not normally limit the rate of synthesis of α or β chains; rather, the two mRNAs compete for some non-specific rate-limiting component of chain initiation.     

定点突变人α_(99)、β_(82)位的血红蛋白及在大肠杆菌中的表达

模拟分析发现血红蛋白两条α珠蛋白链上 99位的 Lys突变为 Cys后 ,它们之间可以形成二硫键 ,两条β珠蛋白链上 82位的 Lys突变为 Cys后可以增加血红蛋白四聚体间氢键的作用 ,分别起到稳定四聚体的作用 .利用寡核苷酸介导的定点突变技术将α99、β82 位的 Lys突变为 Cys.将突变后的血红蛋白插入 p BV2 2 0载体 ,在大肠杆菌中获得了高效表达 ,其表达产物达细菌总蛋白的2 0 %左右 ,并经 Western印迹证实     

Studies on temperature-dependent ultraviolet light-sensitive mutants of bacteriophage T4: the structural gene for T4 endonuclease V.

Mutants of bacteriophage T4 which exhibit increased sensitivity to ultraviolet radiation specifically at high temperature were isolated after mutagenesis with hydroxylamine. At 42 °C the mutants are twice as sensitive to ultraviolet light as T4D, whereas at 30 °C they exhibit survival curves almost identical to that of the wild-type strain. Complementation tests revealed that the mutants possess temperature-sensitive mutations in the v gene.Evidence is presented to show that T4 endonuclease V produced by the mutants is more thermolabile than the enzyme of the wild-type. (1) Extracts of cells infected with the mutants were capable of excising pyrimidine dimers from ultraviolet irradiated T4 DNA at 30 °C, but no selective release of dimers was induced at 42 °C. (2) Endonuclease V produced by the mutant was inactivated more rapidly than was the enzyme from T4D-infected cells when the purified enzymes were incubated in a buffer at 42 °C. From these results it is evident that the v gene is the structural gene for T4 endonuclease V, which plays an essential role in the excision-repair of ultraviolet light-damaged DNA.The time of action of the repair endonuclease was determined by using the mutant. Survival of a temperature-sensitive v mutant, exposed to ultraviolet light, increased when infected cells were incubated at 30 °C for at least ten minutes and then transferred to 42 °C. It appears that repair of DNA proceeds during an early stage of phage development.     

Isolation of mouse FM3A cell mutants with thermolabile thymidylate synthetase by resistance to aphidicolin

Of 625 aphidicolin-resistant clones selected at 33.5°C from mutagenized mouse FM3A cells, 13 clones could not grow at 39.5°C. Five of these clones, chosen at random, resumed growth at 39.5°C when thymidine was added to the culture medium. In hybrids, conditional thymidine auxotrophy was a recessive trait, but aphidicolin-resistance was either a codominant or recessive one depending on the mutant clone used.Thymidylate synthetase activity in crude extracts of these mutants was completely inactivated by preincubation for 30 min at 42°C, whereas that of the parent cells was not affected by the same treatment. Thus, the temperature-sensitive growth of the mutants described here seems to be due to this heat-sensitive thymidylate synthetase.