DNA AND CELL BIOLOGY Volume 11, Number 8, 1992 Mary Ann Liebert, Inc., Publishers Pp. 627-636

Sequences of

DNA

Fragments Contacting the Nuclear

Lamina In Vivo

R. CHRISTOVA, I. BACH,* and Z.

GALCHEVA-GARGOVAÎ

ABSTRACT sequences contacting the nuclear lamina (NL) in vivo, Ehrlich ascites tumor cells were The NL was purified, and the DNA fragments covalently linked to the lamina proteins in UV-irradiated. vivo were cloned and sequenced. Although heterogeneous in length and composition, the sequences displayed homology to the introns and/or flanking regions of different genes, suggesting that functionally distinct regions are organized in a topologically defined manner at the nuclear periphery.

To

study the DNA

INTRODUCTION possible way to address the problem of the correlation between the functional and the topological of DNA in the eukaryotic nucleus is to define the specificity of DNA sequences in relation to their compartmentalization. The nuclear lamina (NL) is a fibrous structure interposed between the chromatin and the inner nuclear membrane (for review, see Gerace, 1986; Newport and Forbes, 1987). It is now accepted that the proteins forming this structure, the nuclear lamins, play important roles in reformation of the nuclear envelope after mitosis (Burke and Gerace, 1986), and in the postmitotic reorganization of chromatin and the intranuclear architecture (Benavente and Krohne, 1986). Recently, we have developed a simple procedure for the purification of the DNA fragments associated with the NL in vivo (Christova and Galcheva-Gargova, 1990). Ehrlich ascites tumor (EAT) cells are first UV-irradiated to crosslink DNA to proteins and thus to avoid the high in vitro affinity of the lamina proteins to DNA, which may lead to artifactual complexes in the course of cell fractionation (Galcheva-Gargova and Dessev, 1987). Next, the NL is isolated and purified so that the structures are free from any associated DNA, except the fragments that were close enough to be cross-linked to it in vivo (NL-DNA). Using this approach, some information about NL-DNA was recently obtained (Galcheva-Gargova, 1988; Christova et ai, 1989). The results suggested that the DNA fragments asso-

One organization

ciated with the NL should be heterogeneous in length and composition. In this study, we present the sequences and a computer analysis of 27 randomly chosen NL-DNA clones.

MATERIALS AND METHODS General The strain of Ehrlich ascites tumor cells

was

maintained

by weekly intraperitoneal injections of 0.20 ml of ascites

fluid into adult mice BALB/c. The cells were harvested on the 7th day and were UV-irradiated in suspension as previously described (Galcheva-Gargova and Dessev, 1987).

Purification of NL-DNA Previously, we described the details of the procedure developed to purify the NL as an intact structure free from any DNA fragments, except those that were initially covalently linked to it by the energy of the UV light (Galcheva-Gargova and Dessev, 1987). The major steps comprise the isolation of chromatin in the presence of EDTA, the digestion with DNase II and RNase, and treatment with 2 M NaCl. Nonequilibrium metrizamide gradients were used to set a density barrier for the bulk of DNA and proteins and to yield at the bottom fraction structures representing nuclear shells of extremely high purity (in the irradiated samples about 2% of the input DNA was sedimented with the lamins) (Galcheva-Gargova and Dessev,

Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. •Oncogenic Viruses Unit, Department of Biotechnology, Pasteur Institute, 75724 Paris, Cedex 15, tPresent address: Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545. Institute of Molecular

627

France.

CHRISTOVA ET AL.

628

ously described (Galcheva-Gargova and Dessev, 1987). The DNA fragments initially cross-linked to the NL were

1987). After proteinase K (50 /ig/ml) digestion of the obtained NL in the presence of 1% NaDodSO« and 1 M NaCl for 3 hr at 37°C and subsequent triple deproteinization with phenol/chloroform/isoamylalcohol (1:1:0,05), NLDNA fragments were precipitated from the aqueous phase with ethanol.

obtained and treated as described in Materials and Methods for cloning in the pBS vector. The sequences of 27 randomly chosen different clones are presented in Table 1. The lengths of the sequenced clones ranged between 113 bp (clone 17) and 474 bp (clone 4). The sequences were first analyzed by a computer program for their nucleotide composition. Only one clone (clone 27) was found to be GC-rich, while about one-third of the rest were AT-rich (Table 2). When checked by the appropriate program, only one clone (clone 8) revealed an open reading frame (ORF). To get some information about the functional characteristics of the NL-DNA sequences, the clones were matched against the sequences in the GeneBank. The obtained homologies are summarized in Table 3. The most striking result was that all sequences (except the clone with the ORF) displayed homologies to the nontranslated introns and/or flanking regions of different genes. More than one-half of the sequences were homologous to different types of repeated sequences. The B1/B2 short interspersed repeats, which comprise up to 6% of total rodent DNA (Kariya et ai, 1987), were most abundant, and were found in more than one-third of the analyzed clones. Different degrees of homology to the long interspersed repeats (L repeats, comprising the R family), representing about 10% of the whole genome (Furano et ai, 1988) were also found. Simple sequences of the type n(GT), n(CCTT), and (TCC) were represented in the population of the sequenced clones as well, clones 7 and 21, respectively. The results summarized in Table 3 are in agreement with the data recently obtained by the C0t analysis of the NL-

Cloning and sequencing of NL-DNA The Bluescribe (pBS) vector was used for cloning in the Sma I site. As the NL-DNA fragments were obtained after DNase II digestion, before ligation they had to be first treated with CIP (Boehringer) for dephosphorylation of the 3' ends, and they were next phosphorylated with T4polynucleotide kinase (Amersham). The possible 5' protruding ends of the fragments were filled up with DNA polymerase I (Klenow enzyme, Amersham) and the possible 3' protruding ends were filled up with T4 polymerase (Amersham). All reactions were performed as described (Maniatis et ai, 1989) using 5 fig of purified NL-DNA

fragments as a starting amount. Competent DH5a cells (BRL) were transformed and white colonies were selected. A rapid, small-scale isolation protocol of plasmid DNA preparation was used to select positive clones with different lengths of inserted DNA fragments. The large-scale preparation and purification of 27 different positive clones was next performed using cesium chloride-ethidium bromide centrifugation (Maniatis et ai, 1989). Double-strand sequencing in both orientations using [a-"S]dATP (Amersham) with the Ml3 forward and reverse primers was performed using the United States Biochemical Sequenase Kit. The sequences obtained were computer analyzed and matches for homologies were searched using the GenBank Data Base (release 69). As controls, the sequences were randomly shuffled by a computer and analyzed using the same program for search of homology.

DNA fragments (Christova et ai, 1989), suggesting that all classes of DNA should be present in the population of sequenced clones. A consensus sequence or a specific motif typical for all or some of the NL-DNA sequences was not found, but the homology of all clones tested to the nontranslated regions of different genes may represent their major characteristic. It is worth noting that the homologies are restricted mainly to the introns and/or flanking regions of some housekeeping genes, genes of the immunoglobulin family, and the different classes of genes of MHC (major

RESULTS

histocompatibility complex).

EAT cells were first UV-irradiated in vivo and the NL was isolated and purified to a very high degree, as previTable 1.

Sequences

a

of

Furthermore, the same sequences when shuffled showed homology lower than 50% to the sequences present in

NL-DNA Fragments

Clone 1 1 51 101 151 201 251 301 351 401

TCACAATTTC AGAAAATGGA ATAAAATCTG ACTTTTTATT TAGCATCTAT

AAGTACCCAC GTGCTCAAGT CGCGCTAAAC TGAATATGAA GACTAGCACA GCATATTATC ATTAATGAGG GGGGTTTGGT GTTGAAGAGA TGTTATCAAA GAGACAGGAA CT

ATAGTCTGTT AATCAATATA TGAGTTGTAT TTGAAAGAAA TGGTAGTGAT CAATCAACTG GCTCTAAATT AGCATGGGCA

AGAAACAAAA AAGCTCACTT AATATGGTTG ATGCATGTGT CCAAGACTTT TACATAAACA AGTGGATACT ACTTTATAAT

TACTTTAATG TAAAAAAAAG CTGCTCAAGC TACTGATATA GTGTGTAATA ACCTGGCTTA GTACAGGACG GAAATCACTT

Clone 2 1

51

GATTTTTTTT TTTTTAAATA AAGACAAAAG GAAGGTGCTG TGGCTCAGGA GTTAAGGCAC TGGTATCACG CCTGGTATTC AGTCCCAGAA CCCAACTGCT

629

DNA CONTACTING THE NUCLEAR LAMINA IN VIVO

101 151 2 01 2 51 301 351

GAAGATTGTC CTCTGTCACC ACAAGGGAAC CATATAAATA AATACATCGA AATAAATAAT ACACAAAAAT AAGTATCTTT TTGGTAATTA TCAGAGAGAC

TGCTCTGTAA TTAAAGGATG CACTACACGC CTTTAATCCC AGACTTGGGA GGCAGAGGCA GGTGGATTTC TGAATTTGAG GCCAGTCTGG TCTAAAGAGT GTGAGTTCCA GGACAGCCAG GGCTACACAG AGAAACCCTG TCTTGAAAAA CAAAAAGAAT ACAAAATTTT TAAAAAAGTG CA

Clone 3 1

51 101 151 201 251 301 351 401

TTAATGGCTC ATGGGAGTGT GGAATTCCAC CACCATCATA TAACATTATT AGTCTTCTAT AATTCTCAGA GCCCTACTTG TCTTGTTTCA

CAGCTGCAAA TGTAGCAGAG GATGGCCTTG GTAGACATAA AGGCCCTTGG TTCTGGGAAG GCTCAATGCC CCAGTGTAGG AGTGGGGAGT CAGGAGTGGG TGAGTGGGTG GGTGGGGGAG GAAGCACAAT AAATAATCAA TCTAATAACC GTTCAGAGGA GTAGTAATAA TATCACAAAA TGATTTGAAT GCTGTTTAAC GGGAAAACCA TTGACAACAT CAGAAGATAT TTAAAAATAT GTTTTGTAAG AATGTGGAAG TTTGCAGAGA TGTACAAGAA ATTCATTGTT GAAGATAGAC TTATGTTGAG CATGTCAACA AACAGGCCAT CAAAGTAATG

CGTACCACTG ATATGGGAAT GAAGACACTG GGTGCCATTC

TCTTCCATGC ATAACATTTG ATGCCCAGGA TCTCTGTGTG TTCTTCTTAA AAGTACATAT TGTAAATCTT GACTGGAACA TTGGGTXATC ACGGAAGGAA

Clone 4 1 51 101 151 201 251 301 351 401 4 51

CATCGTGTTG TCACTAAAAC GGTGGTAGGA AAAGAAAGGA TAACACCTTC CTGAGGAAGA

TCCTACTAGG GAGCTGGGTG GGCTGTCTCG AGTTCACTCA GGTACAAATA TGTTTTAATC TGGATATTTG TATGCAAATA AAAATAAAAA AAAGA

AAGGCCCATG AACGCACTTG TCTCTGCTCA TTGAAGTCTT GCTTCAGTTG TGATTAAGCA

CTGCTTGGAT TCTGAGAAGT TCAGAGATTT TTTCAACACT

TTTGCTTTTT CAGTTATAGG AAGGAGAAGG GAAAAAGTTA AGGAAAGAAG AATAAAATAA AAAAGGGGGA GCCTGGGGCC

Clone 5 1 51 101 151 2 01 2 51 Clone 6 1 51 101

GGAGATCTGA TACATCAAGA TAACAAGAGG GACAAGCTCT AGTGAGTGCA GGTAGCACTA GAGATTACTA GATGGCAAGA AAGAAATATA GCAAGAGAAA CCAAGTTACT TCGGATCATC TCCCATTATA CGAAGTCCTG GATACCCCAC CAACCAGAAC ATCAATAAAC CCAAAAACTG GTGCTTTGAA AAAAGTGAAC AACCCTTAGC CAGAATCACC AGAGGACACA GAGAC

AGTCCGAGAT GGAAAGAACC ATACCAGTTC TATACAAAGA AAGATAGATA

AAAAAGCTTA TCTTTGAAAT GAACCACAGA ATAAGCATCA TATATTTTTT GGAGTACTTT GCGACTTGGA AGGGATATTT TCTTCTTTTT CCCAAGGCXG AXTTGGTCAA AXXXAATATT GTTAC

Clone 7 1

51 101 151

TATGTATTTG TCTGTCTGTG TGAGGCTTAG GACTTTAAGT

CTTTATGTAG TGTGTGTGTG TGTGTGTGTG TGTCTGTCTG

TGTCTATTTC TCTGTGTGTC TCTGTGTGGT ATCAAACATG ATATAAGCTT CATTGAATAT TTCAATGGAT ATTTCAAGCT CAATTTAGTA TTTTATCGAG CTCGAA

Clone 8 1

51 101

GATGTGAATG CTTGTGCCTT ATCTCAAGTA ATGTCCTTGT TGCTCTCTGA GCAGAGAAGT GATGGGCAGC TTCTTCCCAT GGGTGATTTT TGGAAAAGGA CTATCAATTT ACAAATGAAT GCTTGTGAGX TTTTTTTTTT TGGA

Clone 9 51 101

TTCAGAAGAG CAGTCGGGTG CTCTTACCCA CTGAGCCATC TCACCAGCCC CTTAATAATT TTCATTGCTA ATAATTTATA TTCCCTAGTT TTTGAGGTGT CATTCAAACT CCTAATAGGA TCATTTGAAC TTG

Clone 10 1 51 101

CTCGCCAGCC CCTTTGTTTT TTTGGTTTTT TTTTTTTTTT TTTTTTTTTA ATCTTTCGTG ACAGGGTTTT TCTGTGTAGC CCTGGCTATC CTGGAACTCA CTCTTTAGAC CAGGCTGGCC TCAAAG

1

Clone 11 1

51 101

GATTTCAAAC ACCCATTGCC CTGTCTCCCA TCCCTGAGGT TGGAATTATA GCCAAACTAC TATGCATACC TGAAGCATAA GATAATGACG CTTTCAAGTA ACTCTCCCCA CTCCTCTTTC TTACATATGA ATCACATTAT ATATTGAGCC

CHRISTOVA ET AL.

630 151 201 251

AACTCCACAG CATATGCAGG AGGACTTTCT TTCCCTTGCC CGCTCATATT TCATTTCTTC CACTTTCCAA TTTCCAGTTA TTTTTCCTCC TTCTAGAACA ACAAGCAC

Clone 12 1 51 101 151 201

ACTTAAGAGG GTAAATCTTG AGCCTCAGAA AGGAGATCCT TGTCCTCTGA

CCTAGATCCC GCACTCCTTC GCTCTCACGA GTCCCAAACA CCTTCTGGTA

TTATAAAGCT XAAAAAAGAC GTCGTCAACT AGGTGAAAGG CATACAATGG

AGACTTGGTC ATATGTTTCT GGGAGGCAGA GAAAAAGTGA TGGTATATAC AGCTGTGAAC CAAGGATCAA TACACAAGGC CATGTACATA CCTACACTC

Clone 13 1 51 101 151 201

TAATATTCCC AGATAGTAAG CAGGGCCCAC CCCACGCCCT AAACATTTCT

ACCAACAGTG TGGTTATGAG ATAACAGCTC CTTCTGGCCT AAACATGAGA

TGTAAGAGTT CACTTCTTCC ACAACTATCT CTGTGGGCAA

TCTTTTTTTA GGGCTTTGAA AGAGGATCTG GGTCAGTTCT GTGGCTCCAG TTCTGGGGAC TGCATACATA CATGCAGGCA

1 51 101 151 201

CCATCAAAAC CTGCACCACT TTAGTTTATT CCGACAGTAC TCAGGTCTGA

AAGCATTAAA TTGAAGTACT ATTTTACTGT TTTTTCTTCC CAGCAAACAC

ATGATCTAAA GAGCTGGGCT GAGTGTGCAC AGCATTGAGT CCTTACCCAC

TCAAGAACCA TCTGAAGCAA GTGTGTACCA TTCCCGATCA TGAGTCATCT

AGCAGCTAAG CATGATCACC CGGAGATAAC ACTGAGGCTG

Clone 15 1 51 101 151 2 01

AACCTGAATG AGAAGACTTT CTCCTTGCCC CTGCTGGTAC CAAGCTTTTC

TCTTGGAGAG GAAAACATTA TCATTAGATT AGACACACCG AGAGCAGCCA

TCTTTACAGA GCCAACTTCC TTCCATCCCG CAACAGATCG GTTAATTAAT

TGAGCTGCAA AGAATAACCC GTGCCACTCT TCTGCTTGCC GTATGTC

CAAAGGGTTC TGACCTTTTG CTTTAGGCTG TTTCTTAGAT

Clone 16 1 51 101

TAGGGAGGAA GCTGACAGGC AAGAGAGATC AAAGGTGGGG TTTTTGCTTA TGGGTCCAGC TCTCCAAACC CAATGTTTTG GTGACCTGTG ATGGCTATGA TATCTACTAA TATGTGTCCX TTAGACATCT TGTCCAAAC

Clone 17 1 51 101

TAACTATTGC TGAAGCACAG CTCAAAAACC AATCCGTGCT AAGGTGTGGA TTGTGAATTA GGCCCAAATT CCGCATGTGA TTATGGTGGA TCAGTCTCCA GGCTGCCAGA GCT

Clone 18 1 51 101 151 201 251 301

TTTACATATC ATCCTTTCAA TTGGCGCATT CAAGAGCTAG TGTTTGAGCT ACTAAATCAA TCTATTCCGC

TTAAGAAAAT CTTTT

Clone 14

Clone 19 1 51

101 151 Clone 20 1 51

101 151 2 01 251

TAAGGTTAAT CATAGGCATA AAAGATGTTT AAATGACTGA

AAAAAACAAA CACAGCAGCC ACTTTCCAGA GTCTGTGGCA AGGGGAAATG TCCACTCCCG

AGCACACCAC TGTGTTTCCC ATGCTCTCTC AGGGCTTTTC CCATGATCTG GATGCATATT TGTGAGCCAT CATG

CAAGCAAGCC AAAGACTTAG CACCCCATAA CACTAGGAGC

AGCCCTGTAT AGCCCCTGGC TGGCTTCGAC CACGGGTATT ACGAGAGCTT

ACACCTTCTC AGAATAAGTG ACTTGAGGGA CAATGTTATC TCGCTGACCC TCCCAGTACT GCTGATAATC CTGATTGGCA TGCAAAGGAG AGAGAGTTCA GGGTTTGGAT GAACAAGACG GCTTCCTATG TGCTACACTA CTTGTCAGCT TTTGATTC

TCATTTTTGC CCACCCCACA GGAGGGGGGG TAGTGAATCT AGTATGTTTG TGAACAG

TTGCCTATAT GTGCAACACC TTGAGTCTAT CCCCACTGCC CTCATATCAA GAGGAAAGAG GAAGGAAAAG AACAGGGGGA AGGAAGGATA CAGAAGGGGG AGGAGAGATT TGTAGATGGC GGGTGTGACA TCACAGAAAA GCCATCTATG TTTTTTTTTT CTCCATGGGA CAAGGAATCA GAGAAGGCTG CTTCCTTCTC

631

DNA CONTACTING THE NUCLEAR LAMINA IN VIVO Clone 21 1 51 101 151 2 01

CTTCCTTCCT TCCTCTCTTC TGGAGACCTG TCCCTTCTAG TTGGGTCCTC

Clone 22 1 51 101 151

GGATGAGGTG ACCAACACAC CCTAGTGTAA GGATGAGGTC ACCAACACAC CTTAGTGTAA GGATGAGGTA TAAGATCTGT AGAGGGGATT GGATGCCTTG TGCTTCCTCT GCAGAGGTGA GTAGAAGCAA CGTCTTAGAA GAGTCCCAAA GCTCAATG

Clone 23 1 51

101 151 201

TCCTTCCTTC CTTGGGTCTT GCAATTACAC ATGTCAATGG CTGAATGGG

CTTCCTTCCT AGGAGCTAAG CAGACCCTAA ACAGGAACCT

TCCTTCCTTC CTTGATAAAT CTCAATCTGT AACCAGTGAT

GATTTGACAT TCCAACATGG CTTGTTCAGC TGGCTTTCTT GGATCATCAG CTCAGGGATA GACTACCCCA CAATGAGCTG CATAACTACG GATTAAGAAA ATGTCTTACT AGCTGGGCAT ACCTTCAATC CCCCACACTC AGGAGGCAAA GCAGGCAGAC GAGACTAGCC TAGTCTACAG AGAA

CTTCCTCCCT CACAAAGTCC TCGTCTTCTT TAATGTGACC

ATAAAATTGG GACCCTCCCC GATGGCGTAC CTCTGAGTTT

Clone 24 1

GCAGATCTCT AAGGCTTAGC TGCCAGGAGC ATAGCCTGCT TGCTTAGTTG

51 101 151

CCAAGAACCT GAGAGGACTG TCTCAAAGGA AAAGGATGGA TGGATGGCTC CTAAGAGATG ATAGCTTGAA TTGACCTCTG ACCATCAGCA GCAAGCACAG ACGCACACAC ACAAGCTCTG CCACAGA

Clone 25 1

51 101 151

GACCAGCCCT TGTTCTTTCT CGTCCATGGG GTAGGTCCTA

ATGTGGATCC AGAGCCACCT GCTAGGGTTA TGTAGCCTAG

CACACCTTTT GATACAAGTG TAGCCCTAGC GACCTCAAAT CAGAGATCTA CCTGCCTCTG AGAGTGXACA CCAAGACACC CTCCTTTGAG GTTAGC

Clone 26 1 51 101

GATTTTTAAC TGCATGTCTT TATTTCCATC ATTCAAGAGG CAGAGGTGTT TAGAACTGTG TCTCTGTGAG ATAGAGGAAG GCAGGCAGGC AGGCAGGCAG GCAGGCAGGC AGGCAGGCAG GCAGGCCTGA TCTAGG

Clone 27 1 51 101 151

GCTCTCCGGG CTTGGAGCCC ACTTGACAAG ATCACCGGGT

CGAGAACGGT AGCTCXCTGT GAAGAGGGCA CGAGGGGACA

the GenBank lower than 50% (results not shown). It is also important to note that the homologous nucleotides in the shuffled sequences were dispersed throughout the whole length of each clone. In contrast in the original NL clones, "blocks" or big pieces of the sequence were found homologous to a variety of flanking regions and/or introns of different genes.

DISCUSSION The NL is a component of the different types of residual nuclear structures and it has been proposed that it may serve

GGTCTATACT GGACCAAGAC ACGAGAAGGG CTCCAGTTTA

as an

GCGGGCCGAA CCGCACGGTG TCCCTGAACT AGACCCATGA CGTGGGCGAT GCCCGCAACA ACTGGGTA

attachment site for chromatin

Laemmli, 1982). The loops

are

loops (Lebkovski and generated by periodic at-

tachment of the chromatin fiber to a residual nuclear structure called the matrix, scaffold, or cage (Gasser and Laemmli, 1986). Depending on the procedure used, the residual nuclear structure may involve a peripheral lamina, an internal network, and a residual nucleolus. Some evidence exists concerning the scaffold or matrix attachment regions (SAR or MAR). All experimental data concerning the MAR or SAR have been obtained in hybridization experiments of the DNA fragments, cosedimented with the residual structures probed with different parts of a variety of genes. The different sites were not homologous in se-

632

CHRISTOVA ET AL.

Table 2. Nucleotlde Composition of the Sequences the NL-DNA Fragments

pairs

Type A T (%)

402 382 430 475 285 125 186 144 133 126 258 249 235 240 237 139 113 334 158 257 219 158 224 177 176 136 188

66 61 60 60 59 68 64 62 61 60 59 56 56 56 55 55 55 54 54 53 52 52 52 50 49 48 40

Base

Clone number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Total

mouse

were found generally similar in that they are approximately 200-bp-long and AT-rich (Cockerill and Garrard, 1986). All of these recent observations seem to Dominating support the existence of specific nuclear matrix attachment base (%) points at the ends of DNA domains containing particular genes or in the linkers that separate the coding sequences. A 36

A A A A

58.1%

Table 3. Homologies

Clone number

41 36 44

34 35 47 33 A 32 T 32 A 30 T 30 T 29 A 27 A 29 T 28 A 29 T 34 A 30 A 30 A 30 C27 G 34 G 33

DNA

(Kit, 1961)

35 33 32

T 29.3%

of the

of

quence, but

In addition, it has been suggested that the nuclear matrix is involved in the process of DNA replication (Vogelstein et ai, 1980) and transcription (Cook, 1989). The average size of a looped domain is quite similar to the size estimated for a replicón (Rasin et ai, 1986), and the MAR were found near the replication initiation sites (Dijkwel and

Hamlin, 1988). The approach used in our study was a different one. A DNA fraction, cross-linked to the NL in vivo, was cloned, sequenced, and analyzed. The main features of 27 randomly chosen clones may be summarized as follows: (i) a consensus

to

sequence motif present in all

or most

of clones

found; and (ii) all sequences displayed homology the introns and/or flanking regions of several different

was

not

types of genes. A general feature of the DNA fragments associated with the residual structures appears to be the presence of repetitive DNA sequences, previously reported for EAT cells (Kit, 1961). It was suggested by others that the attachment sites may be permanent or transient; the transient sites are thought to be due to replication and transcription, possibly repair. UV-irradiation of EAT cells, a nonsynchronized cell population, would possibly generate a covalent binding to all different types of attachment sites present at the nuclear periphery. It is also possible that the UV light would create binding sites as a result of the reparation processes in transcriptionally active DNA (Mc Cready and Cook, 1984; Mullenders et ai, 1988). As we were not able to find homologies of the analyzed NL-

NL-DNA Clones

to the

Homologies (> 70%) to introns and/or flanking regions

Sequences

ln the

GenBank

Repetitive sequences and other characteristics

Rat insulin-like growth factor II (93.3% in 15 bp) Rat 7-crystallin gene cluster

(70.3% in 37 bp) Lactate dehydrogenase (87.4% in 143 bp) IGE receptor

(89.1% in 140) Receptor of granulocyte colony-stimulating factor (84.4% in 154 bp) Glycogen phosphorylase (73.6% in 159 bp) Serum amyloid P component (77.3% in 132 bp) 4.5S rRNA repetitive sequences (73.1% in 150 bp) Prolactin gene

(76.7% in 163 bp)

Bl repetitive sequences (91.3% in 126 bp)

Bl and B2 repetitive sequences in the different parts of the clone

DNA CONTACTING THE NUCLEAR LAMINA IN VIVO Table 3.

Clone number

633

(Continued)

Homologies (> 70%) to introns and/or flanking regions Human hypoxanthine phosphoribosyltransferase (93.8% in 16 bp)

Repetitive seguences and other characteristics

Alu

repetitive

sequence

Human adenosine deaminase gene (100% in 15 bp) T-cell receptor (76% in 25 bp)

Nicotinic acetylcholine receptor

(75.9%

in 29

bp)

T-cell receptor (82.2% in 107 bp)

LI

Rat

long interspersed repetitive sequences (84% in 106 bp) /3-Globin complex (78.3% in 23 bp)

repetitive element (LINE-1) (LIRn) —specific member of

LINE 3 LINE-1 Bl

repetitive

sequence

0-Casein gene (72.4% in 29 bp)

Human factor XIII b subunit (100% in 16 bp) MHC (H-2) S region (76.5% in 102 bp)

Simple

(GT)ll

sequences -

Ig germ-line x V-region (77.3% in 88 bp)

10

Interleukin (78.9% in 85 bp) Rabbit /3-like globin cluster (70.8% in 48 bp) Rat tyrosine aminotransferase (78.9% in 71 bp) Rat gene for growth hormone (75% in 92 bp) B2 associated protein (80.5% in 77 bp) LI repetitive sequence (98% in 50 bp) Renin (Ren-2-d) (93.2% in 52 bp) Tumor rejection antigen P815A (84% in 125 bp) LT lymphotoxin (LT) (87.5% in 104 bp)

Tumor necrosis factor (87.5% in 104 bp)

11

ORF

Direct repeat (RU) B2

repetitive element

LINE-1

repetitive

sequence

A /«-like

repetitive

sequences

Bl

repetitive

sequences

B2

repetitive

sequence

(TNF)

Rearranged erythropoietin (83% in 112 bp) 0-Globin complex (76.5% in 34 bp) Rat 7-crystallin cluster (75% in 28 bp) Human interleukin-2 14 bp)

(92.9% in 12

Rat steroid hydroxylase IIA1 (94.1% in 17 bp)

T-cell

activating protein (77.1% in82bp)

I gene for MHC class II

(74.2% in 31 bp) Rat long interspersed repetitive (77.8% in 27 bp)

sequence

LINE 3

(specific member

of

LINE-1)

CHRISTOVA ET AL.

634 Table 3.

(Continued)

13

Ins DNA (cellular component that mediates integration and excision of polyoma virus DNA) (74% in 104 bp) Erythropoietin (76.1% in 119 bp) Rat serine dehydratase (70% in 100 bp)

14

Rat TM-4 gene for fibroblast tropomyosin 4 (71.7% in 53 bp) Human migration inhibitory factor related protein 8 (MRP8) (100% in 13 bp) T-cell receptor a/5 chain locus (88.9% in 18 bp)

15

7-crystallin gene cluster (74.2% in 31 bp; 100% in 14 bp) Human prothymosin-alpha in 20 bp)

17

pseudogene

A lu

repetitive

sequence

T-cell receptor a/ö chain locus (81.5% in 27 bp) Rat 7-crystallin gene cluster (70% in 40 bp)

Opsin gene (MOPS) (100% in 13 bp) Rat kallikrein-binding protein (93.3% in 15 bp) Rat cytochrome P450 (94.45 in 18 bp) Human adenosine deaminase

18

(74.1% in 27 bp) Low-affinity IgE receptor (70% in 50 bp) Human hypoxanthine phosphoriboxyltransferase (85.5% in 26 bp) /3-Globin complex (90% in 20 bp)

19

(94.1% in 17 bp) Rat cytochrome P-452 (73.2% in 41 bp) Human large fibroblast proteoglycan (100% in 14 bp) i3-Globin complex (81% in 21 bp)

Rat

Rat

A /«-like retrosposon Alu

repetitive

sequence

7-crystallin cluster

7-crystallin cluster

in 14 bp) Rat ornithine decarboxylase (81% in 21 bp)

(100% 20

other characteristics

Rat

(100% 16

Repetitive seguences and

Homologies (> 70%) to introns and/or flanking regions

Clone number

/3-Glucuronidase (94.4% in 18 bp)

pseudogene

Human serine protease (100% in 14 bp) Human al-microglobulin gene (70% in 87 bp) Human factor IX gene

(81% in26bp)

B1/B2

repetitive

sequence

Alu repetitive sequence

DNA CONTACTING THE NUCLEAR LAMINA IN VIVO

Table 3.

Clone number 21

22

23

635

(Continued)

Homologies (> 70%) to introns and/or flanking regions Cardiac myosin heavy chain (95.2% in 63 bp) MHC class II (95.2% in 62 bp) Rat carboxypeptidase B (96.8% in 63 bp) MHC class I (95.2% in 63 bp) Rat cytochrome P450 (76.9% in 26 bp) T-cell receptor a/ô (90% in 17 bp) Human T-cell receptor complex (94% in 16 bp) Rat TRPM-2 gene (100% in 14 bp) T-cell receptor a/5 chain (78% in 112 bp) Nucleolin gene (82% in 93 bp)

Glycerophosphate dehydrogenase (80% in 92 bp) Rat DNA for Bl repeat 85 bp)

Repetitive seguences and other characteristics

Polypyrimidine repeat Polypyrimidine repeat B2

repetitive sequence

B2

repetitive

B1/B2

sequence

repetitive element

Bl

repetitive element

Bl

repetitive element

(84% in 24

Human 7-B-crystallin (90% in 20 bp) Human plasminogen activator inhib.-l (79% in 38 bp)

25

U6 small nuclear RNA (74% in 72 bp)

B1/B2

a-Fetoprotein (70% in 118 bp) /3-Globin complex (95% in 19 bp)

Bl

repetitive element

Bl

repetitive element

MHC class II H2-IE ß (95% in 58 bp) T-cell surface antigen (87.6% in 125 bp) Human cytomegalovirus (94% in 16 bp) T-cell receptor (72% in 28 bp) MHC class II la region (76% in 73 bp)

Simple

sequence

(AGGC)„

Simple

sequence

(AGGC)„

26

27

The

repetitive

sequences

origin of the listed homologous sequences is indicated except for the mouse ones.

DNA clones to some coding sequences (Table 3), most probably those complexes are not formed at the nuclear periphery as frequently as in the nuclear interior. Because of the higher abundance of the introns and/or flanking regions in the available data in the GenBank, we may not fully rule out the possibility of a random binding of the

NL to DNA. But this seems quite improbable because the ratio of noncoding versus coding sequences in the analyzed clones was very high (26:1). Further, assuming that it was possible during the ligation reaction to prepare clones composed of more than one binding sequence, the above ratio would be even higher.

CHRISTOVA ET AL.

636

One must note the similarity of the features of the íase domain of Chinese hamster ovary cells. Mol. Cell. Biol. 8, 5398-5409. MAR/SAR and of the analyzed NL-DNA sequences. This was to be expected as the nuclear lamina is an element of FURANO, A., ROBB, S., and ROBB, F. (1988). The structure of the regulatory region of the rat LI (LIRn, long interspersed rethe different matrix and scaffold structures. But the appeated) DNA family of transposable elements. Nucleic Acids proach used in our study is inconsistent with some possible Res. 16, 9215-9231. artefacts due to cosedimentation and redistribution of GALCHEVA-GARGOVA, Z. (1988). In Metabolism and EnzyDNA fragments during the isolation procedure (Cook, J. Nucleic Acids Including Gene

mology of

1988).

of the sequences of the parts the nuclear lamina in vivo was an attempt to reveal a nonrandom organization of chromatin at the nuclear periphery and the possible role of the lamina structure in DNA organization. Concerning the logical question of the type of genes positioned at the nuclear periphery in the different species, experiments analyzing NLDNA should be performed with cells and cell lines different from EAT cells. In summary,

of DNA

our

analysis

contacting

ACKNOWLEDGMENTS

Zelinka and J. Balan, eds. 287.

(Plenum Press,

Manipulation. York) pp. 283-

New

GALCHEVA-GARGOVA, Z., and DESSEV, G. (1987). Crosslinking of DNA to nuclear lamina proteins by UV irradiation in vivo. J. Cell Biochem. 34, 163-168. GASSER, S., and LAEMMLI, U. (1986). The organization of chromatin loops: Characterization of a scaffold attachment site. EMBO J. 5, 511-518. GERACE, L. (1986). Nuclear lamina and organization of nuclear architecture. Trends Biol. Sei. 11, 443-446. KARIYA, Y., KATO, K., HAYASHIZAKI, Y., HIMENO, S., TARUI, S., and MATSUBARA, K. (1987). Revision of consensus sequences of human Alu repeats —a review. Gene 51, 1-10.

We thank Dr. Bernard Gaudron for his help in computer analysis of the sequences. We are grateful to Prof. M. Yaniv and Dr. S. Cereghini for valuable discussions. I. Bach was supported by a Boehringer Ingelhein Fonds fellowship, and Z. Galcheva-Gargova by fellowships from CNRS and FEBS.

REFERENCES BENAVENTE, R., and KROHNE, G. (1986). Involvement of nuclear lamins in post mitotic reorganization of chromatin as demonstrated by microinjection of lamin antibodies. J. Cell. Biol. 103, 1847-1854. BURKE, B., and GERACE, L. (1986). A cell free system to study reassembly of the nuclear envelope at the end of mitosis. Cell 44, 639-652. CHRISTOVA, R., and GALCHEVA-GARGOVA, Z. (1990). A simple method for isolation of DNA fragments associated with the nuclear lamina in vivo. J. Biochem. Bioph. Meth. 20, 107111.

CHRISTOVA, R., YANEVA, J., and GALCHEVA-GARGOVA, Z. (1989). Some features of DNA fragments associated in vivo with the nuclear lamina. Biosci. Rep. 9, 587-592. COCKERILL, P., and GARRARD, W. (1986). Chromosomal

loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell 44, 273-282. COOK, P. (1988). The nucleoskeleton: Artefact, passive framework or active site. J. Cell Sei. 90, 1-6. COOK, P. (1989). The nucleoskeleton and the topology of transcription. Eur. J. Biochem. 185, 487-501. DIJKWEL, P., and HAMLIN, J. (1988). Matrix attachment regions are positioned near replication initiation sites, genes, and an interamplicon junction in the amplified dihidrofolate reduc-

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NEWPORT, I., and FORBES, D. (1987). The nucleus: Structure, function, and dynamics. Annu. Rev. Biochem. 56, 535-565. RASIN, S., KEKELIDZE, M., LUKANIDIN, E., SCHERRER, K., and GEORGIEV, G. (1986). Replication orgins are attached to the nuclear skeleton. Nucleic Acids Res. 14, 8189-8207.

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Address reprint requests to: Dr. Z. Galcheva-Gargova Worcester Foundation for Experimental Biology 222 Maple A venue Shrewsbury, MA 01545 Received for

publication May 4, 1992; accepted July 10,

1992.