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The globin gene family represents an attractive system for the study of gene regulation during mammalian development, as its expression is subject to both tissue-specific and temporal regulation. While many aspects of globin gene structure and expression have been described extensively, relatively little is known about the cis-acting DNA sequences involved in the developmental regulation of globin gene expression. To begin to experimentally define these regulatory sequences, we have taken the approach of introducing cloned globin genes into the mouse germ line and examining their expression in the resulting transgenic animals. Here we describe a series of transgenic mice carrying a hybrid mouse/human adult beta-globin gene, several of which express the gene exclusively or predominantly in erythroid tissues. These studies demonstrate that regulatory sequences closely linked to the beta-globin gene are sufficient to specify a correct pattern of tissue-specific expression in a developing mouse, when the gene is integrated at a subset of foreign chromosomal positions.

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Partial digestion of rat liver nuclei with staphylococcal nuclease results in the liberation of nucleoprotein complexes consisting of one or more v bodies. By velocity centrifugation we have isolated the monomeric subunit in relatively pure form. We find that this subunit contains 185 base pairs of DNA and 240,000 daltons of protein, resulting in a protein to DNA ratio identical to that of unperturbed chromatin. The isolated monomeric particle is further susceptible to internal nuclease attack resulting in the solubilization of 46% of the monomeric DNA. Analysis of the resistant DNA reveals a complex but highly reproducible pattern of DNA fragments ranging from 160 to 60 base pairs in length. Analysis of the reassociation kinetics of the isolated subunit DNA reveals that most, if not all genomic sequences, are involved in this basic subunit structure. No special frequency class of DNA is absent from v bodies. Furthermore, virtually all liver sequences transcribed into mRNA are present in v body DNA. These results indicate that v body formation may be random with respect to DNA sequence and suggest that the mere presence of v bodies over a specific regioni of DNA is not sufficient to restrict its transcription.Over the past year an experimentally consistent model of chromatin structure has emerged which postulates the presence of regularly repeating nucleoprotein subunits joined by short segments of DNA. Biochemical evidence for a periodic protein-DNA complex was initially introduced by Hewish and Burgoyne (1) and subsequently confirmed in several laboratories (2-6). These data demonstrate that mild nuclease digestion of nuclei results in the liberation of a series of nucleoprotein particles, v bodies, containing DNA fragments whose molecular weights are all multiples of a unit fragment 150-200 base pairs in length. Subsequent studies (4,5,7,8) have revealed that the v body represents a transient intermediate in the digestion process and that cleavage of DNA within the P body results in the generation of a true limit digest reflecting the internal structure of the monomeric subunit.Electron microscopic observations (9, 10) are in accord with the biochemical data and reveal the existence of spherical nucleoprotein particles which are joined by a short filament of DNA. These particles can be observed closely apposed to one another in gently disrupted nuclei, providing convincing evidence that these nucleoprotein particles reflect a level of structure characteristic of the chromatin fiber in vio.The elucidation of a subunit structure in chromatin immediately poses the question as to the possible role of this structure in those biological processes in which chromatin participates. In this report we describe procedures for the isolation and characterization of highly purified v bodies.We find that most if not all of the DNA sequences in the genome are involved in the formation of these particles. No specific frequency class of DNA is restricted from particle formation. Further, virtually all sequences express...

Rabbit chromosomal DNA contains a cluster of four linked beta-like globin genes arranged in the orientation 5'-beta 4-(8kb)-beta 3-(5 kb)-beta 2-(7-kb)-beta 1-3'. Determination of the nucleotide sequence of gene beta 1 confirms that this gene corresponds to the second type of two common co-dominant alleles encoding the adult beta-globin chain. With the exception of two nucleotide substitutions in the large intervening sequence (intron), the intron and flanking sequences are identical with the nucleotide sequence of the first type determined by Weissmann et al. (1979). A 14S polyadenylated transcript containing large intron sequences (possibly a mRNA precursor) is detected in the bone marrow cells of anemic rabbits. Gene beta 2 has limited sequence homology to adult and embryonic beta-globin probes and lacks a detectable mRNA transcript in the erythropoietic tissues examined. It contains at least one intervening sequence analogous to the large intron in gene beta 1. Genes beta 3 and beta 4 both contain an intron of 0.8 kb. Partial DNA sequence analysis indicates that the large intron in beta 4 is located between codons for amino acids lysine and leucine in an analogous position to that of the large intron in beta 1. In addition, a second smaller intron interrupts the 5' coding sequences of gene beta 4. Both genes beta 3 and beta 4 are transcribed in embryonic globin-producing cells. Their DNA sequence homology is limited, however, to a segment of approximately 0.2 kb located on the 5' side of the large intron.

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