The asynchrony of gamma- and beta-chain synthesis during human erythroid cell maturation. III. gamma- and beta-mRNA in immature and mature erythroid clones

The synthesis of the β-crystallin polypeptides has been studied in different regions of the embryonic chicken lens. Seven β-crystallin polypeptides ranging in molecular weight from approximately 19,000 (19K) to 35,000 (35K) daltons were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Each polypeptide was synthesized in a rabbit reticulocyte cell-free system supplemented with RNA from the embryonic lens fiber cells suggesting that each is encoded by a separate mRNA. Analysis of the cell-free translation products of the RNAs from 6-, 15-, and 19-day-old embryonic chicken lens fibers demonstrated that all seven polypeptides are translated at each of the stages and that the proportion of β-crystallin mRNAs increases as the chicken embryo matures. Fingerprints of methionine-containing tryptic peptides indicated that the three predominant β-crystallin polypeptides synthesized in the reticulocyte lysate (20K, 26K, and 35K) have related but distinct primary structures. Surprisingly, both the 35K β-crystallin polypeptide and its mRNA were selectively absent from the cells in the central region of the epithelium. Synthesis of this polypeptide from extracted RNAs was detected in the elongating cells of the equatorial region of the epithelium and from the fiber cells. In contrast to the 35K polypeptide, the six lower-molecular-weight β-crystallin polypeptides were synthesized in a reticulocyte lysate directed by RNAs extracted from all three regions of the lens. These data indicate that lens cell elongation and fiber cell differentiation in the embryonic chicken are accompanied by the appearance of the mRNA for the 35K polypeptide.     

Role of gene order in developmental control of human gamma- and beta-globin gene expression.

To determine the effect of gene order on globin gene developmental regulation, we produced transgenic mice containing two tandemly arranged gamma- or beta-globin or gamma beta- and beta gamma-globin genes linked to a 2.5-kb cassette containing sequences of the locus control region (LCR). Analysis of constructs containing two identical gamma or beta genes assessed the effect of gene order on globin gene expression, while analysis of constructs containing tandemly arranged gamma and beta genes assessed any additional effects of the trans-acting environment. When two gamma genes were tandemly linked to the LCR, expression from the proximal gamma gene was three- to fourfold higher than expression from the distal gamma gene, and the ratio of proximal to distal gene expression remained unchanged throughout development. Similarly, when two beta genes were tandemly linked to the LCR, the proximal beta gene was predominantly expressed throughout development. These results indicate that proximity to LCR increases gene expression, perhaps by influencing the frequency of interaction between the LCR and globin gene promoters. An arrangement where the gamma gene was proximal and the beta gene distal to the LCR resulted in predominant gamma-gene expression in the embryo. When the order was reversed and the gamma gene was placed distally to the LCR, gamma-gene expression in the embryo was still up to threefold higher than expression of the LCR-proximal beta gene. These findings suggest that the embryonic trans-acting environment interacts preferentially with the gamma genes irrespective of their order or proximity to the LCR. We conclude that promoter competition rather than gene order plays the major role in globin gene switching.     

Assembly of gamma- with alpha-globin chains to form human fetal hemoglobin in vitro and in vivo

Soluble gamma-globin chains were expressed in bacteria and purified to assess the mechanism of gamma- and alpha-chain assembly to form Hb F. Formation of Hb F in vitro following incubation of equimolar mixtures of gamma and alpha chains was about 4 x 10(5)-fold slower than assembly of alpha and beta chains to form Hb A in vitro. Results of assembly for gamma(116Ile-->His) and gamma(112Thr-->Asp) chains with alpha chains were similar to that of beta chains, whereas assembly of gamma(112Thr-->Cys) and alpha chains was similar to wild type gamma chains, indicating that amino acid differences at alpha1beta1 and alpha1gamma1 interaction sites between gamma116 Ile and beta116 His are responsible for the different assembly rates in vitro in the formation of Hb F and Hb A. Homoassembly in vitro of individual gamma chains as assessed by size-exclusion chromatography shows that gamma and gamma(112Thr-->Cys) chains form stable dimers like alphabeta and alphagamma that do not dissociate readily into monomers like beta chains. In contrast, gamma(116Ile-->His) chains form monomers and dimers upon dilution. These results are consistent with the slower assembly rate in vitro of gamma and gamma(112Thr-->Cys) with alpha chains, whereas the faster rate of assembly of gamma(116Ile-->His) and gamma(112Thr-->Asp) chains with alpha chains, like beta chains, may be caused by dissociation to monomers. These results suggest that dissociation of gamma(2) dimers to monomers limits formation of Hb F in vitro. However, yields of soluble Hb F expressed in bacteria were similar to Hb A, and no unassembled alpha and gamma chains were detected. These results indicate that gamma chains assemble in vivo with alpha chains prior to forming stable gamma(2) dimers, possibly binding to alpha chains as partially folded nascent gamma-globin chains prior to release from polyribosomes.     

Simian homologues of human gamma-2 and betaherpesviruses in mandrill and drill monkeys

Recent serological and molecular surveys of different primate species allowed the characterization of several Kaposi's sarcoma-associated herpesvirus (KSHV) homologues in macaques, African green monkeys, chimpanzees, and gorillas. Identification of these new primate rhadinoviruses revealed the existence of two distinct genogroups, called RV1 and RV2. Using a degenerate consensus primer PCR method for the herpesvirus DNA polymerase gene, the presence of KSHV homologues has been investigated in two semi-free-ranging colonies of eight drill (Mandrillus leucophaeus), five mandrill (Mandrillus sphinx), and two hybrid (Mandrillus leucophaeus-Mandrillus sphinx) monkeys, living in Cameroon and Gabon, Central Africa. This search revealed the existence of not only two distinct KSHV homologues, each one belonging to one of the two rhadinovirus genogroups, but also of two new betaherpesvirus sequences, one being close to cytomegaloviruses and the other being related to human herpesviruses 6 and 7 (HHV-6 and -7). The latter viruses are the first simian HHV-6 and -7 homologues identified to date. These data show that mandrill and drill monkeys are the hosts of at least four novel distinct herpesviruses. Moreover, mandrills, like macaques and African green monkeys, harbor also two distinct gamma-2 herpesviruses, thus strongly suggesting that a second gamma-2 herpesvirus, belonging to the RV2 genogroup, may exist in humans.     

Synthesis of tetrazole analogs of gamma- and delta-amino acids.

N-benzyloxycarbonyl-protected alpha- or beta-amino alcohols, easily prepared from alpha- and beta-amino acids, were converted into aldehydes and directly reacted with (triphenyl phosphoranylidene) acetonitrile, leading to unsaturated nitriles. Treatment of nitriles with NaN(3) and ZnBr(2) produced unsaturated gamma- and delta-amino tetrazoles, which were deprotected and converted to the corresponding saturated compounds by catalytic hydrogenation. For the case of delta-amino tetrazole, the methylation of the acidic moiety occurred after treatment with CH(2)N(2), leading to the N(1)- and N(2)-methylated constitutional isomers, which were separated by column chromatography and hydrogenated.