The positions of preferential DNA interaction with the nuclear matrix were mapped within the domain of the chicken a-globin genes in transcriptionally active erythroblast nuclei and inactive nuclei of mature erythrocytes. In the latter, only two major distinct attachment sites were observed, close to the A+T-rich sequences previously found at the boundaries of the domain. Sequencing of these structural matrix attachment points revealed several known DNA motifs; some of them were present on both sides of the domain. In actively transcribing erythroblast nuclei of adult animals, a large fraction of the transcribed area was represented in nuclear matrix DNA, including upstream and downstream elements. In particular, adult aA-and ccD-globin genes were found in matrix DNA, while the transcribed but translationally unexpressed embryonic IT gene was underrepresented. The data are discussed in terms of the existence of stable or structural and expressionrelated matrix attachment sites; correlations to the origin of replication and the units of transcription of the domain are shown.In eucaryotic nuclei, chromatin is arranged in loops composed of 10 to 100 kilobase pairs (kbp) of DNA (for a review, see reference 16). In metaphase chromosomes and in interphase nuclei, these loops are anchored at their bases by interaction with a residual protein framework named the scaffold or nuclear matrix, respectively (1, 3, 28). Recent observations strongly suggest that a functional link exists between the matrix-DNA contact points and the control of the activity of chromosomal domains (1-3, 9, 10, 15-17, 29). Attempts to establish a correlation between the distribution of specific sequences along the DNA chain and the sites of attachment of chromatin loops to the nuclear matrix have been inconclusive (13).We have demonstrated that eucaryotic DNA is systematically punctuated by easily denaturing A+T-rich regions, for which the term A+T-rich linkers (ATRLs) was proposed (27) and which frame some genomic domains, as shown in a-type avian globin genes or the ftz gene in Drosophila melanogaster (20,26). Three different types of A+T-rich DNA segments were identified: (i) isolated ATRLs interspersed in the genome, on average, once every 10 to 40 kbp of DNA; (ii) clusters of ATRLs occurring, on average, once every 25 to 100 kbp of DNA; and (iii) the A+T breaks which subdivide the segments between genes or are placed in intervening sequences (26,27,33). Different roles for individual ATRLs, clusters, and breaks have been tentatively proposed (33). The average distance between ATRLs is equivalent to the lengths of DNA loops (1,13,14,30) and chromosome domains containing transcriptional units as measured by DNase sensitivity (21, 36) and in vivo transcription (6,7,24). Interestingly, some observations demonstrate that nuclear matrix attachment sites of several specific eucaryotic domains are placed within A+T-rich DNA (9, * Corresponding author. t Present address: Engelhardt

The sequence of a DNA fragment about 1 Kbp long located at the 3' boundary of the chicken alpha globin gene domain, including the 3'-side matrix attachment point and the site of transcription termination, was determined. It contains a repetitive DNA element and the AT-rich (easily denaturable) DNA segment conserved at the same position in the duck genome. The repetitive sequence was identified by computer analysis as being a member of the CR1 family. Within the non-repetitive part of the AT-rich DNA fragment, four topoisomerase II recognition sites were found which might be indicative of matrix attachment. Furthermore, two distinct regions were identified, possessing strong homology to a number of noncoding consensus sequences, one of them to a limited part of the LTR of HTLVIII, and the other to the replication origin of Polyoma virus JC. DNA shift experiments showed that the CR1 repeat binds specifically an abundant nuclear protein factor. The binding site for this factor was identified by footprinting and turned out to be closely related to the previously described recognition site for the TGGCA-binding protein, the chicken analog of nuclear factor 1 (NF-1). Finally, the CR1 repeats within the chicken alpha and beta globin gene domains were mapped. All these observations are discussed in terms of the organization of the 5' and 3' boundaries of the functional genomic domains forming a chromatin loop including all avian alpha type globin genes.

Monoclonal antibodies demonstrated high conservation during evolution of a prosomal protein of M(r) 27,000 and differentiation--specific expression of the epitope. More than 90% of the reacting antigen was found as a p27K protein in the free messenger ribonucleoprotein (mRNP) fraction but another protein of M(r) 38,000, which shared protease fingerprint patterns with the p27K polypeptide, was also labelled in the nuclear and polyribosomal fractions. Sequencing of cDNA recombinant clones encoding the p27/38K protein and comparison with another prosomal protein, p30-33K, demonstrated the existence of a common characteristic sequence pattern containing three highly conserved segments. The genes Hs PROS-27 and Hs PROS-30 were mapped to chromosomes 14 (14q13) and 11 (11p15.1), respectively. The structure of the p27K protein shows multiple potential phosphorylation sites, an NTP-binding fold and an RNA-binding consensus sequence. The Hs PROS-27/beta-galactosidase fusion protein binds a single RNA of about 120 nucleotides from total HeLa cell RNA. Sequence comparisons show that the Hs PROS-27 and Hs PROS-30 genes belong to the gene family that encodes the prosome--MCP (multicatalytic proteinase)--proteasome proteins. Comparison with other members of the family from various species allowed us to show that the tripartite consensus sequence characteristic of the alpha-type sub-family is conserved from archeobacteria to man. The members of this gene family are characterised by very high evolutionary conservation of amino acid sequences of homologous genes and 20%-35% sequence similarity, between different family member within the same species and are clearly distinct from the beta-type family.

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