A single amino acid substitution in v-erbB confers a thermolabile phenotype to ts167 avian erythroblastosis virus-transformed erythroid cells.

A library of recombinant bacteriophage was prepared from ts167 avian erythroblastosis virus-transformed erythroid precursor cells (HD6), and integrated proviruses from three distinct genomic loci were isolated. A subclone of one of these proviruses (pAEV1) was shown to confer temperature-sensitive release from transformation of erythroid precursor cells in vitro. The predicted amino acid sequence of the v-erbB polypeptide from the mutant had a single amino acid change when compared with the wild-type parental virus. When the wild-type amino acid was introduced into the temperature-sensitive avian erythroblastosis virus provirus in pAEV1, all erythroid clones produced in vitro were phenotypically wild type. The mutation is a change from a histidine to an aspartic acid in the temperature-sensitive v-erbB polypeptide. It is located in the center of the tyrosine-specific protein kinase domain and corresponds to amino acid position 826 of the human epidermal growth factor receptor sequence.

Replacement variant histone genes contain intervening sequences.

The nucleotide sequences of two chicken histone genes encoding replacement variant H3.3 polypeptides are described. Unlike the replication variant genes of chickens (and almost all other organisms), these genes contain intervening sequences; introns are present in both genes in the 5' noncoding and coding sequences. Furthermore, the replacement variant histone mRNAs are post-transcriptionally polyadenylated. The locations, but not the sizes, of the two introns within the coding segments of the two genes have been exactly conserved, whereas the intron positions in their respective 5' flanking regions differ. Although both H3.3 genes predict the identical histone polypeptide sequence, they are as different from one another as each of them is from a more common replication variant H3.2 gene in silent base substitutions within the coding sequences. Thus, the H3.3 polypeptide sequence has been precisely maintained over a great evolutionary period, suggesting that this class of histones performs a strongly selected biological function. Although replacement variant histones can account for more than 50% of the total H3 protein in the nuclei of specific chicken tissues, the steady-state level of H3.3 mRNA is nearly the same (and is quite low) in all tissues and ages of animals examined. These properties suggest novel mechanisms for the control of the basal histone biosynthesis which takes place outside of the S phase of the cell cycle.

Developmental regulation of beta-globin gene switching.

A chicken erythroid cell-specific enhancer is located in the intergenic region between the adult beta- and embryonic epsilon-globin genes. In this paper we show that the beta-globin enhancer stimulates transcription of both genes. epsilon-Globin is, however, inappropriately regulated since it is expressed in both embryonic and adult red blood cells. Appropriate stage-specific regulation is observed for both genes when they are present on one plasmid. By analysis of deletion and substitution mutants, we conclude that beta-globin tissue- and developmental stage-specific regulation is mediated by interaction of the beta-globin enhancer with a positive regulatory element within the adult beta-globin promoter, the developmental stage selector element (SSE).

A 3' enhancer is required for temporal and tissue-specific transcriptional activation of the chicken adult beta-globin gene.

The chicken adult beta-globin gene is one of the more intensively investigated developmentally regulated loci in higher eukaryotes. Detailed molecular analysis of the locus allows precise examination of the chromosomal changes that occur on activation of the gene during erythroid maturation. The best studied of these changes are the acquisition of DNase I hypersensitivity, developmentally correlated alteration of CpG-specific cytosine methylation patterns and in vitro assembly of erythroid-specific protein complexes 5' to the gene that mimics in vivo creation of the 5' DNase I hypersensitive 'region' lying 60 to 260 nucleotides 5' to the beta-globin cap site in red blood cell chromatin. Here we demonstrate that proximal beta-globin DNA sequences lying greater than 112 base pairs (bp) 5' to the cap site are not involved in determining the erythroid-specific induction characteristics of this gene in transient expression assays, whereas an enhancer sequence within a 300-bp PvuII fragment lying approximately 400 nucleotides 3' to the polyadenylation signal is intimately involved in determining the erythroid cell specificity and correct time of induction of beta-globin transcription during red cell maturation.

The nucleotide sequence of the embryonic chicken beta-type globin genes.

The complete nucleotide sequence is reported for both of the embryonic chicken beta-type globin genes, rho and epsilon. These two genes lie at the 5' end and 3' end, respectively, of the four closely linked chicken beta-type globin genes relative to the direction of transcription. Both genes have structures that are typical of functional beta-type globin genes in that they contain two intervening sequences with the 5'-most intron being relatively small (108 base pairs in both) and the 3' intron being large (541 base pairs in rho and 973 base pairs in epsilon). Both embryonic genes contain consensus flanking sequences which are similar to those previously found in the adult chicken beta-globin gene and in a variety of other genes transcribed by RNA polymerase II. The rho- and epsilon-globin gene sequences are very similar from approximately nucleotide -130 (with the mRNA initiation site as +1) to the end of exon II. Noncoding sequences outside this region (e.g. intron II and DNA 5' to -130) are highly divergent. This may indicate that the DNA sequence elements which specifically activate embryonic beta-type globin gene expression in primitive red cells are contained within the -130 to +468 region. Comparison of the overall sequences of rho and epsilon to that of beta strongly supports the hypothesis that the embryonic avian beta-type globin genes diverged from the adult beta-gene by a process separate from that which was responsible for the generation of nonadult mammalian beta-type globin genes.