In chicken leukemia cells globin genes are fully transcribed but their rnas are retained in the perinucleolar area.

Using hybridization in situ with a ribo-probe recognizing transcripts of the chicken alpha A globin gene, we show here that in proliferating AEV-transformed erythroblasts this gene is strongly transcribed, but the corresponding transcripts are retained in the nuclei. Most surprisingly, this globin RNA accumulates in the perinucleolar areas in a pattern never observed before. Upon induction of cells to differentiate, leading to productive expression of the hemoglobins, the transcripts of the alpha A globin gene were found for the most part in the cytoplasm. In the nuclei of differentiated cells, the globin RNA is concentrated in one or two specific spots, which are likely to represent the "processing centers" (PCs) of the globin RNA. The results presented indicate that posttranscriptional steps of regulation involving in particular the perinuclear areas are of major importance for erythroid differentiation.

Prosomes form sarcomere-like banding patterns in skeletal, cardiac, and smooth muscle cells.

Prosomes (20S proteasomes) constitute the catalytic core of the 26S proteasomes, but were first observed as factors associated with unstranslated mRNA. Recently, their RNase activity was discovered together with the fact that their proteolytic function is dispensable in adapted human cells. By indirect immunofluorescence using monoclonal antibodies, we demonstrate as a general phenomenon, regular intercalation of specific types of prosomes into the sarcomeric structure of all types of striated muscle. Surprisingly, in cultured smooth muscle cells without sarcomeric organization, some prosomes also form regular striations in extended projections of cytoplasmic regions. The significance of their sarcomeric distribution is not understood as yet, but the pattern we observe is very similar to that shown by others for muscle-specific mRNAs, identified by in situ hybridization, and that of the cognate proteins. A role of prosomes in the cotranslational assembly of the myofibrillar proteins is suggested, since prosomes organize into pseudo-sarcomeric patterns prior to formation de novo of the actin-myosin arrangement.

Non-clonability correlates with genomic instability: a case study of a unique DNA region.

Instability of eukaryotic DNA in constructs propagated in prokaryotic hosts is a frequently observed phenomenon. With the exception of a very high A+T-content and the presence of multiple repetitions, no general rule at the basis of this phenomenon is actually known. The intergenic spacer located between the pi and alpha(D) chicken alpha-type globin genes is frequently deleted from recombinant phages and plasmids. Here we have cloned this DNA fragment using a specially designed bacterial strain (SURE competent cells, Stratogene). Comparative analysis of DNA of recombinant clones bearing deletions and clones containing the intact genomic DNA fragment has revealed two important DNA sequence motifs that contribute to the unclonability of eukaryotic DNA in prokaryotic cells. First, the similarity to bacterial transposons (i.e. the presence of repeats flanking a several kilobase DNA fragment) may cause the loss of the fragment during propagation of the recombinant DNA in E. coli. Second, a high content of rotationally correlated kinkable elements (TG*CA steps) may result in non-clonability of the DNA sequence. Interestingly, the latter type of "unclonable" DNA sequence motifs identified in the globin gene domain is unstable (frequently rearranged) also in the eukaryotic chromosome resulting in a local polymorphism. In the chicken domain of alpha globin genes this unstable DNA sequence seems to be partially protected by interaction with nuclear matrix proteins.

Exon/intron organisation of human proteasome PROS-27 K gene.

The exon/intron structure of the human proteasome PROS-27 gene was established by means of partial sequencing of its genomic clones and comparison with the chromosome 14 sequences from the data bases. The gene contains seven exons spanning over 19kb. Introns of the gene contain numerous Alu type repeats, Mer 2 and LINE type repeats. Pattern of the repeats indicates conservatism of the sequence.

Specific types of prosomes distribute differentially between intermediate and actin filaments in epithelial, fibroblastic and muscle cells.

First observed as components of non-translated mRNP complexes, prosomes harbour RNase and several proteinase activities; they are also the central constituent of the "Multicatalytic Proteinase (MCP) complexes" or "26S-proteasomes". In two recent publications (Arcangeletti et al., 1997b; De Conto et al., 1997) we have shown, by applying a new fixation technique, that these particles distribute differentially between the cytoskeletal networks of intermediate filament (IF) and actin types; previously they had been observed exclusively on the intermediate filaments. Here we further investigate the distribution of prosomes of several types, distinct by their subunit composition, between the IF of vimentin type and the actin network, as well as in the 3D space of the cell. It is shown that subtypes of prosomes occupy specific networks of the cytoskeleton, and that this pattern is specific for a given cell type. Confocal microscopy shows that prosome cytodistribution is not homogeneous in the 3D space: in the perinuclear area they colocalize most strongly with the IF, and more peripherally with the microfilament/stress fiber system; connections may exist between the two networks. Furthermore, new data indicate that the prosome-actin interaction may participate in the molecular structure of the stress fibers.

Extensive methylation of a part of the CpG island located 3.0-4.5 kbp upstream to the chicken alpha-globin gene cluster may contribute to silencing the globin genes in non-erythroid cells.

Here, we show that in the chicken genome, the domain of alpha-globin genes is preceded by a CpG island of which the downstream part ( approximately 0.65 kbp) is heavily methylated in lymphoid cells; it is either non-methylated or undermethylated in erythroid cells. Recombinant plasmids were constructed with the corresponding DNA fragment (called "uCpG") placed upstream to a reporter CAT gene expressed from the promoter of the alpha(D) chicken globin gene. Selective methylation of CpG dinucleotides within the uCpG fragment suppressed fivefold the expression of the CAT gene, when neither this gene itself nor the alpha(D) promoter were methylated. Methylation of CpG dinucleotides within the alpha(D) gene promoter did not modify the suppression effect exerted by methylated uCpG. We interpret these results within the frame of the hypothesis postulating, that methylation of the upstream CpG island of the chicken alpha-globin gene domain may play an essential role in silencing the alpha-globin genes in non-erythroid cells.

In mouse myoblasts nuclear prosomes are associated with the nuclear matrix and accumulate preferentially in the perinucleolar areas.

Prosomes are the core of 26S proteasomes, although they were originally observed as 20S particles associated with cytoplasmic mRNPs. Here we show for the first time that prosomes are also genuine constituents of the nuclear matrix, chromatin and the nuclear RNP networks. Using mouse myoblasts we tested three monoclonal antibodies recognising the prosomal subunits p23K, p27K and p30K, and found that the corresponding prosome subclasses are characterised by a variable distribution pattern within the nuclei. Their presence on the nuclear matrix, and most abundantly in the perinucleolar area, is of particular importance. When myoblasts fuse into myotubes, the distribution pattern of certain types of prosomes on the nuclear matrix changes drastically. Surprisingly, DNA strongly interferes with the detection of prosomal antigens by immunofluorescence methods, whereas RNA, histones and other proteins soluble in 2 M NaCl have no such effect. This 'masking' of prosomes can be completely overcome by extensive or even mild digestion with DNase I or restriction enzymes. Many nuclear prosomes can be solubilized by combined treatment with 0.5% Triton X-100 and 2 M NaCl, and others can be released by digestion of DNA and/or RNA, and about 10-20% of nuclear prosomes remain tightly bound to the protein-based nuclear matrix.

A novel gene is transcribed in the chicken alpha-globin gene domain in the direction opposite to the globin genes.

A novel gene transcribed in the direction opposite to that of the globin genes was found in the chicken alpha-globin gene domain. Northern hybridisation with single-stranded riboprobes revealed that a 4.5-kb poly(A)+ RNA is transcribed in antisense polarity with respect to the globin genes. The transcription unit encoding this RNA seems to overlap the entire cluster of alpha-globin genes and extends at least 15 kb upstream from pi, the first of the alpha-globin genes. This new transcript shows partial sequence homology with that encoded by the human "-14" gene. An oligonucleotide based on part of a restriction fragment of chicken DNA that is 80% homologous to exon 4 of the human "-14" gene hybridises with a 4.5-kb RNA molecule. In situ hybridisation of globin-antisense probes, that detect polyribosomal mRNAs of 1.7 and 2.5 kb on Northern blots, shows these "antisense" transcripts to be present in the cytoplasm. The 4.5-kb RNA is absent in polyribosomal poly(A)+ RNA and may, hence, represent a nuclear pre-mRNA transcribed from the chicken gene that is homologous to the human "-14" gene. The expression of this gene is not specific to erythroid cells; analogous transcripts were also detected in poly(A)+ RNA extracted from a chicken lymphoblastoma cell line (HP50). Taken together, these data allow us to postulate the existence in the chicken genome of a novel gene, for which we suggest the name "ggPRX" in analogy to the murine mProx1, a gene identified in the upstream region of the alpha-globin gene domain in mice.

In the nucleus and cytoplasm of chicken erythroleukemic cells, prosomes containing the p23K subunit are found in centers of globin (pre-)mRNA processing and accumulation.

Prosomes were originally identified as 20S particles associated with untranslated mRNA; they also constitute the core of the 26S proteasomes. The cellular distribution of three types of prosomes characterized by the presence of subunits with molecular masses of 23, 27, and 30 kDa was analyzed using an immunocytochemical approach on cultured chicken erythroblasts. The prosomes containing the p27K and p30K subunits were found in diffuse distribution in both nuclei and cytoplasm. In contrast, the prosomes containing the p23K subunit, although relatively rare in the nuclear space, were found concentrated in one or two large spots. Using in situ hybridization with an alpha(A)-globin gene-specific riboprobe we found that the p23K-type prosomes colocalize in the nucleus with centers of globin (pre-)mRNA processing, and of mRNA accumulation in the cytoplasm. This result suggests there is local coincidence of specific-type prosome function with processing and, possibly, transport of a particular kind of (pre-)mRNA.

Functional analysis of DNA sequences located within a cluster of DNase I hypersensitive sites colocalizing with a MAR element at the upstream border of the chicken alpha-globin gene domain.

We have cloned and sequenced a genomic DNA fragment of chicken containing a cluster of DNase I hypersensitive sites (DHS) located 11-15 kb upstream from the first gene of the alpha-globin gene domain and including a constitutive DHS flanked by two erythroid-specific ones. A 1.2-kb subfragment of the DNA fragment under study located upstream to the constitutive DHS and colocalizing roughly with one of the erythroid-specific DHS was shown to possess the properties of a matrix association region (MAR). The cloned DNA sequences were tested for their ability to serve as promoters and/or influence transcription from the promoter of the alphaD globin gene. In the region studied, we did not find any promoters or enhancers that were active in erythroid cells. The whole DNase I hypersensitive region and some of its subfragments showed a silencing effect when placed downstream from the reporter gene. The expression of the reporter gene was completely abolished, however, when these DNA fragments were placed between the alphaD promoter and the reporter gene. Thus, they seem to act as transcription "terminators." Numerous polyadenylation signals (AATAAA) and an AT-rich palindrome were found within the sequenced DNA fragment. These observations are discussed within the frame of the hypothesis postulating that continuous transcription is essential for maintaining the active status of genomic domains. Furthermore, it is suggested that the DNA fragment studied contains a negative control element that keeps globin genes silent within the chromatin domain permanently open in nonerythroid cells.

Dynamic distribution and formation of a para-sarcomeric banding pattern of prosomes during myogenic differentiation of satellite cells in vitro.

Myogenesis proceeds by fusion of proliferating myoblasts into myotubes under the control of various transcription factors. In adult skeletal muscle, myogenic stem cells are represented by the satellite cells which can be cultured and differentiate in vitro. This system was used to investigate the subcellular distribution of a particular type of prosomes at different steps of the myogenic process. Prosomes constitute the MCP core of the 26S proteasomes but were first observed as subcomplexes of the untranslated mRNPs; recently, their RNase activity was discovered. A monoclonal antibody raised against the p27K subunit showed that the p27K subunit-specific prosomes move transiently into the nucleus prior to the onset of myoblast fusion into myotubes; this represents possibly one of the first signs of myoblast switching into the differentiation pathway. Prior to fusion, the prosomes containing the p27K subunit return to the cytoplasm, where they align with the gradually formed lengthwise-running desmin-type intermediate filaments and the microfilaments, co-localizing finally with the actin bundles. The prosomes progressively form discontinuous punctate structures which eventually develop a pseudo-sarcomeric banding pattern. In myotubes just formed in vitro, the formation of this pattern seems to preceed that produced by the muscle-specific sarcomeric (alpha)-actin. Interestingly, this pattern of prosomes of myotubes in terminal in vitro differentiation was very similar to that of prosomes observed in vivo in foetal and adult muscle. These observations are discussed in relation to molecular myogenesis and prosome/proteasome function.

Increased prosomal proteins in breast cancer cells and in neighboring normal cells in Parsi and non-Parsi populations.

Monoclonal antibodies were raised against the prosomal proteins p27K, p29K and the prosome-like protein p21K (PLP) from normal breast glandular cells and from benign and malignant tumors. They were used to clarify the involvement of prosomes in tumorigenesis of human breast cells. Immunostaining showed the distribution of prosomes in the cytoplasm and nuclei of cells from European normal women (EN) and Parsi (P) and non-Parsi (NP) benign (B) and malignant (M) tissues. The flow-cytometry studies showed an increased mean percentage of labeled cells, particularly with anti-p27K prosomal protein mAb, in malignant tissue from NP compared to EN. The p21K data indicated an increase in the number of cells labeled by flow-cytometry studies in all groups compared to EN, while p29K-expressing cells were more abundant in NPN, PB, PM and NPM. Intergroup comparison showed that the mean percentage of cells labeled with anti-p27K and anti-p29K was significantly higher in PB than in NPB, as seen by flow cytometry, whereas there was a higher production or accumulation of the p21K (PLP) prosomal protein in NPM than in PM, as seen by immunostaining. By comparison with EN, there were also significantly more normal cells containing the three antigens in the apparently normal tissue in the neighborhood of the tumor in NPM, and more cells containing p21K in PM patients than in EN. As prosomes are involved in the cell differentiation and in the cell cycle control, the changes observed in breast tissues may be related to oncogenic processes. Furthermore, the modified subunit pattern of prosomes in cancer and, possibly, pre-cancer tissue may be of interest for diagnosis purposes.

Modification of cytoskeleton and prosome networks in relation to protein synthesis in influenza A virus-infected LLC-MK2 cells.

Modifications of the cytoskeleton and protein synthesis were investigated in LLC-MK2 cells during infection by FPV/Ulster 73, an avian strain of influenza A virus. During infection, the cytoskeleton and the prosome networks undergo a dramatic reorganization, which seems to be at least temporally differentiated for each cytoskeletal system, i.e. microfilaments (MFs), microtubules (MTs), intermediate filaments (IFs). In order to evaluate the role of the three different cytoskeletal networks during FPV/Ulster infection, studies were carried out on cellular and virus-specific protein synthesis and viral production, using drugs which selectively affect individual cytoskeletal systems. Our data show that the perturbation of the IF system, but not that of the MFs or MTs, seems to have a strong inhibitory effect on virus production and cellular and viral protein synthesis. Furthermore, the dynamics of IFs and prosomes were investigated during viral infection and, at no time, dissociation of the prosome and IF networks was observed. Taken together, these results strongly support the idea that the interactions between the protein synthesis machinery, the cytoskeleton, and the prosomes are all affected by viral infection in a partially coordinated manner.

Changes in the subunit distribution of prosomes (MCP-proteasomes) during the differentiation of human leukemic cells.

The subunit composition of cell-internal and surface prosomes during phorbol myristate acetate (PMA)-induced differentiation of human leukemic T lymphocytes (CCRF-CEM cell line) was studied in relation to clusters of differentiation (CD) markers. PMA inhibited cell growth and decreased the amounts of CD1a and CD4 while CD3, CD8, CD25, CD45, CD57 and MHCI increased it; the p53 anti-oncogene increased while actin levels remained constant. Cells incubated with the inducer PMA for 3 days and placed in fresh inhibitor-free medium resumed growth at a low rate, while the CD values slowly reverted to those of the initial phenotype. The presence and relative amounts of prosome subunits were analyzed by flow cytometry, light and fluorescent microscopy and Western blotting using 3 monoclonal antibodies (p25K, p27K and p30-33K MAbs). The decrease in cytoplasmic antigens on day 3 was remarkable (cells followed for 7 days) while increased surface antigens were observed. Changes in the subcellular distributions of prosome antigens, particularly the p25K and p30-33K subunit, were correlated with a partial arrest of the cell cycle. Interestingly, the composition of cell internal and surface prosomes showed different patterns of change.

Visualization of prosomes (MCP-proteasomes), intermediate filament and actin networks by "instantaneous fixation" preserving the cytoskeleton.

A new "instantaneous" fixation/extraction procedure, yielding good preservation of intermediate filaments (IFs) and actin filaments when applied at 37 degrees C, has been explored to reexamine the relationships of the prosomes to the cytoskeleton. Prosomes are protein complexes of variable subunit composition, including occasionally a small RNA, which were originally observed as trans-acting factors in untranslated mRNPs. Constituting also the proteolytic core of the 26S proteasomes, they are also called "multicatalytic proteinase (MCP) complexes" or "20S-Proteasomes." In Triton X-100-extracted epithelial, fibroblastic, and muscle cells, prosome particles were found associated primarily with the IFs (Olink-Coux et al., 1994). Application of "instantaneous fixation" has now led to the new observation that a major fraction of prosome particles, composed of specific sets of subunits, is distributed in variable proportions between the IFs and the microfilament/ stress fiber system in PtK1 epithelial cells and human fibroblasts. Electron microscopy using gold-labeled antibodies confirms this dual localization on classical whole mounts and on cells exposed to instantaneous fixation. In contrast to the resistance of the prosome-IF association, a variable fraction of the prosome particles is released from the actin cytoskeleton by Triton X-100 when applied prior to fixation. Moreover, in vitro copolymerization of prosomes with G-actin made it possible to observe "ladder-like" filamentous structures in the electron microscope, in which the prosome particles, like the "rungs of a ladder," laterally crosslink two or more actin filaments in a regular pattern. These results demonstrate that prosomes are bound in the cell not only to IFs but also to the actin cytoskeleton and, furthermore, not only within large M(r) complexes (possibly mRNPs and/or 26S proteasomes), but also directly, as individual prosome particles.

Prosome cytodistribution relative to desmin and actin filaments in dividing C2.7 myoblasts and during myotube formation in vitro.

Prosomes constitute the multicatalytic proteinase (MCP) core of the 26S proteasomes, but were first observed as subcomplexes of untranslated mRNP; this suggests that they play a putative role in the control of protein biosynthesis in addition to their catabolic enzymatic function. In previous investigations it was shown that some prosomes colocalize with the intermediate filaments (IF) of the cytoskeleton, of the cytokeratin type in epithelial cells, and of the vimentin type in fibroblasts. Studies on adult rat muscle carried out with prosome-specific monoclonal antibodies (p-mAbs) have shown, surprisingly, that specific types of prosomes predominantly occupy a particular zone in between the M and the Z lines of the sarcomeric structure. The data presented here show that the subunit composition of prosomes changes when the dividing C2.7 myoblasts fuse into myotubes. We show furthermore that, in dividing C2.7 myoblasts, prosomes colocalize with the desmin network as well as with that of actin, in a distribution that changes with the subunit pattern of the prosomes investigated by individual p-mAbs. Surprisingly, when myogenic fusion is induced, specific types of prosomes move first to the nuclei; later on, they reappear in the cytoplasm. There, superimposing initially onto the reorganizing desmin filaments that run from one pole of the prefusion myoblast to the other, prosomes gradually colocalize with the actin fibers in the fusing myotubes, finally forming a "pearl on a string" pattern. These results are discussed in relation to parallel observations of prosome distribution between the actin and IF networks not only in epithelial cells but also in fusing muscle satellite cells, which made it possible to monitor the complete buildup of the sarcomeric structure.

Prosomes (proteasomes) changes during differentiation are related to the type of inducer.

The core of the 26S proteasome, the 20S prosome, is a highly organized multi-protein complex found in large amount in malignant cells. Differentiation of several cell lines, including the monoblastic U937 and the lymphoblastoid CCRF-CEM, is accompanied by a general decrease in the prosome concentration when phorbol-myrirtic-acetate (PMA) and retinoic acid plus dihydroxyvitamine D3 (RA+VD) are used. Incubation of U937 cells for three days with PMA or RA+VD causes differentiation, but the resulting patterns of prosome labeling in the cell and on the plasma membrane are not the same. In contrast, the same kind of prosome changes occur in U937 and CCRF-CEM cells when PMA is used as inducer. The intracellular distribution of prosomes is also linked to malignancy and differentiation. Prosomes are found in the nucleus and the cytoplasm of cancer cells; and treatment with RA+VD decreases the prosomes in the nucleus whereas PMA causes various prosome proteins changes. These results indicate that prosomes are important in cell regulation and in the expression of malignancy.

Characterization of prosomes in human lymphocyte subpopulations and their presence as surface antigens.

Prosomes, also called "multicatalytic proteinase" (MCP) or "proteasomes," are a new type of ubiquitous RNP particle present in some archeobacteria and in all eukaryotic cells tested from yeast to human. They were discovered as subcomplexes of untranslated messenger-ribonucleoproteins (mRNP) and later found to have a MCP activity putatively involved in antigen processing. Being composed of variable sets of characteristic proteins and associating small RNAs (pRNA), families of individual "mosaic" prosome particles seem to characterize the differentiation type and physiological state of individual cells and tissues. Here, prosomes from human lymphocytes, isolated and characterized biochemically and by Western blot analysis, were found to differ in their subunit composition compared to other human prosomes. Surprisingly, prosomal antigens were discovered at the outer surface of blood cells monitored by flow cytometry with monoclonal antibodies to individual prosomal proteins. It was observed that human T and B lymphocytes have variable and characteristic prosomal antigens at their surface according to their CD classification. Interestingly, the lymphocyte subpopulations most strongly labeled by the anti-p25K and anti-p27K mAbs were the NK and B cells.