134 SEQUENTIAL ASSESSMENT OF CELL CYCLE S-PHASE IN FLOW CYTOMETRY TO MEASURE LYMPHOCYTE ACTIVATION IN VITRO

Ch. Kohler, M.N. Kolopp-Sarda, A. De March-Kennel, M.C. Béné, G.C. Faure

Laboratoire d'Immunologie, Faculté de Médecine, B.P. 184 - 54505 Vandoeuvre les Nancy Cedex, France

Proper signals of lymphocyte activation induce the proliferation of these cells, both *in vivo* and *in vitro*. This multiplication can be induced *in vitro* by mitogens or specific antigens and is usually measured using methods involving tritiated thymidin. Cellular proliferation can also be analyzed by flow cytometry techniques based on cell cycle analysis through the measurement of DNA content after staining with intercalants or DNA dyes. We applied this method to lymphocytes from 113 individuals to evaluate lymphocyte proliferation after stimulation *in vitro* by a mitogen (Phytohaemagglutinin, PHA) or a recall antigen (tetanus toxoid), using a kinetic approach with 4 points sequential measurements of the S and G₂ phases over 6 days of culture. The proportion of cells in S phase after PHA stimulation was significantly higher than in controls overall and as early as on day 3 of the culture. Activation with a recall antigen induced significantly increasing S-phase cell proportions up to day 6. These data suggest that flow cytometric assessment of the S phase could be a useful alternative to isotopic methods measuring lymphocyte reactivity in vitro.

135 MOLECULAR COMBING: A NEW APPROACH TO MOLECULAR CYTOGENETICS AND PHYSICAL MAPPING

A. Bensimon, X. Michalet, C. Schurra and J. Herrick.

Laboratoire de Biophysique de l'ADN, Institut Pasteur, Paris, France

In a process called "molecular combing" DNA molecules attached at one end to a solid surface are extended and aligned by a receding air-water interface and left to dry on the surface.

It turns out that the force the interface exerts on the DNA is strong enough to extend the molecule but to weak to break the bond between the molecule and the surface. The local action of the interface is the same on each of the molecules in solution: they are identically stretched.

Simple, controlled and reproducible optical mapping on purified DNA molecules, individual sorted chromosomes and total genomic DNA is made possible by applying Fluorescence in situ hybridisation (FISH) to combed DNA. The utility and accuracy of FISH on combed molecules are illustrated by several examples. (i) mapping of lambda DNA restriction fragments along linearized 49 Kbp long lambda phage DNA molecules with 1 Kbp precision, (ii) mapping of cosmid clones along a 470 Kbp yeast artificial chromosome (YAC) with 5 kbp precision and estimation of the gaps and the overlaps between them, and (iii) ultrahigh resolution comparative nucleic acid hybridisation to combed DNA molecules. This has allowed us to study, in a relatively simple manner, chromosomal rearrangements, insertions and deletions of different genetic disorders with high precision.

136 FISH ON CHROMOSOMES: SOME INTRODUCTORY REMARKS

Dr Alain Bernheim

Laboratoire de Cytogénétique et Génétique Oncologiques (URA 1967

CNRS), Institut Gustave Roussy, 36 rue Camille Desmoulins 94805

Villejuif Cedex, France

Morphological cytogenetics has been transformed in molecular cytogenetics by the introduction of FISH techniques. The nucleus content and organisation can be analysed during all the cell cycle, either on condensed chromosome, intact nuclei, section of nuclei, whole DNA, stretched DNA.

The FISH techniques are rapidly expanding with various probes:

- unique DNA segments from few hundred base pair to several megabase;

- repeat sequences for centromeric probes, telomeric allowing even the use of oligonucleotides for PRINS;

- whole or partial chromosome for painting;

- Total DNA for comparative in situ hybridization which allows to demonstrate chromosomal losses or gains from tumoral DNA;

- stretched DNA or chromatin which allows a resolution as low as few kb. Digital imaging by cooled single or color CCD cameras and image processing are more and more used. Recently Schrock et al (Science 1996) used an interferometer to determine the full spectrum of light emitted by each stained chromosome. If a combinatorial approach is used by labeling the painting probes for each chromosome with a different assortment of fluochromes, as few as five dyes can give enough combinations to give one for each human chromosome type.

Among an exponentially growing amount of applications, determination of the very complex chromosomal abnormalities in cancer cells are one which may help to understand the genetic change leading to cancer and may provide roads to therapy.
Index alphabétique

Abboud M. - 71
Abgrall J.F. - 15
Adnet J.J. - 71
Ait El Mkadem S. - 96
Albina E. - 29
Alonso Vega C. - 57
Andréo B. - 85
Andriamihaja M. - 41
Angof F. - 3 - 7 - 9
Antoine E. - 49
Antonioli I. - 120
Arnoulet C. - 114
Assailly J. - 20
Aubry J.P. - 25
Audigier J.C. - 70
Audrêzet M.P. - 15
Autran B. - 92
Auvinet P. - 91
Azgui Z. - 123
Bailly P. - 97
Bakour D. - 115
Balitrand N. - 130
Baloche J.C. - 14
Barbounaki I. - 90
Barddelay C. - 49
Barray S. - 22
Barre P. - 42
Barthelemy C. - 70
Bastard C. - 122
Baudouin F. - 55
Baume D. - 108
Belaud-Rotureau M.A. - 105
Belhomme P. - 3
Belhoussine R. - 33
Belloc F. - 95 - 105
Béné M.C. - 55 - 110 - 111 - 134
Bensimon A. - 135
Bensussan A. - 23
Berger F. - 107
Bergeron M. - 50
Bernard P. - 105
Bernheim A. - 66 - 85 - 90 - 136
Bernier M. - 109 - 111
Berthois Y. - 18 - 19
Bertrand P. - 77
Besson I. - 26 - 28
Bikoue A. - 31
Blaise D. - 114
Blanc C. - 92
Blancher A. - 125
Bloyet D. - 3
Boenders J. - 83
Bonnaud G. - 117
Bonnefoy J.Y. - 25
Bony V. - 97
Bord A. - 24
Bouchara J.Ph. - 44
Bouchu C. - 94
Boudry C. - 9 - 11 - 12 - 16
Boumsell L. - 23
Bourgeois C.A. - 83
Boutonnat J. - 27 - 32 - 100
Broechevelt T. - 109
Brohée D. - 128 - 129
Brugère D. - 49
Brulet P. - 70
Brunet C. - 53 - 54
Bruzzoni-Giovanelli H. - 65
Buffet-Janvresse C. - 35 - 88

Caillot D. - 127
Callat M.P. - 34
Calvo F. - 65
Camoin-Jau L. - 26
Campos L. - 94 - 111
Candelier J.J. - 85 - 90
Cantinieaux B. - 109
Capel P. - 57
Carayon P. - 24
Carcelain G. - 92
Cardot H. - 16
Carli P.M. - 127
Carpenter D. - 36
Carrere F. - 44
Cartron J.-P. - 97
Casanovas O. - 12 - 90
Casasnovas R.O. - 111
Casellas P. - 24
Chabannon C. - 114
Chabasse D. - 44
Chaffanjon P. - 108
Chagnon C. - 108
Chapery M.F. - 41
Chardonnel Y. - 63
Charonov S. - 62
Charrin C. - 111
Charroin L. - 97
Chasse J. - 14
Chassevent A. - 51 - 58
Chatelain B. - 109
Chaumarel I. - 111
Chermant J.L. - 11 - 12
Chevailler A. - 44 - 124
Chomienne C. - 130
Chomik A. - 67
Chouraqui M. - 108
Chretien P. - 115
Clot J. - 132 - 133
Coffe C. - 127
Cohen J. - 45
Coiffier B. - 107
Colin D. - 39
Colin D.A. - 46
Colin M. - 53
Colita D. - 121
Collet L. - 76
Colliot G. - 49
Coninx P. - 69
Conjeaud H. - 34
Coster M. - 11 - 12
Cottier M. - 70
Coullin P. - 85 - 90
D'Anjou J. - 77
D'hautcourt J.L. - 51 - 57 - 58 - 59 - 109
Dastugue N. - 125
Datry T. - 3
Daudet N. - 104
Daveau M. - 101 - 102
Davoust J. - 64
Dayer E. - 120
De Bentzmann S. - 45
De Geyter H. - 57
De March-Kennel A. - 134
Debré P. - 92
Delbeke E. - 108
Delisle M.J. - 69
Delneste Y. - 25
Delobelle A. - 74
Delville J.P. - 57

Deman D. - 3 - 9
Demandolx D. - 64
Demur C. - 125
Deneys V. - 57 - 109
Depermentier F. - 49b
Desplechain C. - 78
Di Paola R. - 63 - 65 - 66
Dieterlen A. - 67
Djamali A. - 133
Dobo I. - 124
Doisy A. - 43
Domy M. - 127
Doublet B. - 79
Doutrelant P. - 74
Drevot P. - 56
Dreyfus F. - 87
Drouet M. - 117
Duchayne E. - 125
Ducrotte P. - 79
Dufer J. - 69 - 103
Duigou F. - 3 - 72 - 75 - 76
Dumain P. - 95
Dumas D. - 4
Dumitrescu A.M. - 121
Duperray C. - 5 - 42
Dupont J.M. - 87 - 91
Dupré J.M. - 88
Dupuy O. - 87 - 91
Durrieu F. - 95 - 105
Dusser C. - 17 - 18 - 19
Dussossay D. - 24
Dutrillaux B. - 83
Duval C. - 77
Duval Ch. - 122
Egloff H. - 78
Elbim C. - 30 - 53 - 118
Elmoataz A. - 7 - 8 - 10
Exbrayat C. - 132
Farcet J.P. - 23
Farkas D.L. - 1
Faucher C. - 114
Faure G. - 111
Faure G.C. - 110 - 134
Favre M. - 111
Féigueux N. - 132
Felman P. - 107
Feneux D. - 84
Férec C. - 15
Feuillard J. - 123
Fichelson S. - 87
Filippi C. - 37
Floch V. - 15
Fontaine M. - 35
Fontenay-Roupie M. - 87
Fradelizi D. - 34
Franck F. - 78
François A. - 79 - 102 - 122
François V. - 88
Frouin F. - 63 - 65 - 66
Fruchart C. - 122
Galand P. - 93
Galiégue S. - 24
Gallais A. - 77
Gane P. - 97
Garand R. - 111
Gastal C. - 54
George F. - 26 - 31 - 54
Germain F. - 43
Girodon F. - 127
Gosset P. - 73 - 74
Goubern M. - 41
Gougerot-Pocidalo M.A. - 30 - 118
Gouin I. - 130
Goujon N. - 78
Guyotat D. - 94
Hamon S. - 42
Hardy E. - 15
Healy J.C. - 96
Hebbache A. - 49b
Hemet J. - 79 - 102
Henry-Amar M. - 76
Herlin P. - 3 - 7 - 8 - 9 - 10 - 11 - 12 - 13 - 14 - 16 - 72 - 75
Herrick J. - 135
Hiron M. - 101
Hurez D. - 124
Husson B. - 109
Israël N. - 118
Jacquey S. - 67
Jaloustre C. - 95
Janossy G. - 31
Jayat-Vignolles C. - 47 - 117
Jbilo O. - 24
Jeanne-Pasquier C. - 76
Jeannin P. - 25
Joret J.B. - 3
Jouen Beades F. - 34 - 119
Julien R. - 47
Jung G. - 67
Kahn E. - 63 - 65 - 66
Kennes B. - 128
Keriel C. - 37
Klein C. - 61
Klijanienko J. - 83
Kohler Ch. - 134
Kolopp-Sarda M.N. - 134
Kroemer G. - 99
Kuhlein E. - 125
Kujas M. - 6
Lacombe F. - 95 - 105
Lacoste L. - 95
Laquerrière A. - 79
Laudier B. - 84
Laurent J.L. - 70
Le Moine P. - 124
Le Roux G. - 123
Le Tessier D. - 87 - 91
Lebarr A. - 87
Lebeau C. - 76
Lebreton J.P. - 101 - 102
Leclerq N. - 76
Lecomte-Houcke M. - 73
Lefebvre J.L. - 74
Lenoir M. - 104
Lenormand B. - 34 - 122
Leonard C. - 84
Leprat P. - 47
Leprêtre S. - 34
Lequeux N. - 80 - 81
Lescoarnec J. - 88
Lesesve J.F. - 34 - 122
Lesty C. - 6
Leverve X. - 37
Liautaud F. - 69
Liautaud-Roger F. - 103
Linares-Cruz G. - 65
Lizard G. - 63
Lorenzato M. - 71
Louarn J. - 42
Lu H. - 48
Lucas L. - 61
Lyoumi S. - 102
Maciorowski Z. - 83
Macro M. - 106
Malcus C. - 53 - 97
Malcus-Vocanson C. - 97
Malet M. - 106
Mandard A.-M. - 3 - 76
Mandy F. - 50
Manfait M. - 33 - 62

Marchand J. - 24
Marion S. - 53
Marnay J. - 76
Martin E. - 20
Martin P.M. - 18
Masson E. - 3
Masson S. - 101 - 102
Maubon I. - 70
Maynadie M. - 111 - 127
Mazerbo F. - 108
Mazzon A.M. - 109
Mendel I. - 88
Menet A. - 35
Menot M.L. - 130
Mercadier A. - 53
Mercier B. - 15
Meunier O. - 39 - 46
Meyer J.J. - 67
Michalet X. - 135
Michel P. - 79
Michels J.-J. - 72 - 76
Miet S. - 26
Mishal Z. - 2
Moiceanu A.D. - 121
Monconduit M. - 122
Monteil H. - 39 - 46
Morjani H. - 33
Moskovtchenko P. - 127
Mosnier-Damet M. - 78
Motoiu A.I. - 121
Mourad G. - 133
Mousseau M. - 27 - 32
Mozziconacci M.J. - 114
Mugneret F. - 111
Muirhead K. - 36
Mutin M. - 31
Necker A. - 114
Nève P. - 128 - 129
Noirot M. - 42
Ohlsson-Wilhelm B. - 36
Olive D. - 114
Osmont A. - 13 - 14
Oziel S. - 114
Pagés I. - 56
Pagés J. - 107
Pagniez M. - 58
Paillasson S. - 40
Palmari J. - 18 - 19
Parešy M. - 79
Paris E. - 119
Paschoud N. - 21
Pelegrini M. - 66
Pellestor F. - 85 - 90
Penel C. - 19
Perek N. - 94
Peries L. - 92
Perroux-Goumy L. - 124
Petit P.X. - 41 - 99
Petitprêtre G. - 24
Philip P.J.M. - 52 - 55 - 108 - 116
Philippe C. - 66
Pignon S. - 127
Pillet S. - 118
Piriou L. - 92
Plancoulaine B. - 3 - 13 - 14
Pléau-Varet J. - 6
Ploton D. - 60 - 61
Poitevin-Later F. - 97
Poncelet P. - 26 - 28 - 31
Portalès P. - 132 - 133
Pouchet M. - 48
Pradier O. - 57 - 109
Praz G. - 120
Preira A. - 118
Prévost G. - 39 - 46
Provot F. - 101 - 102
Puchelle E. - 45
Quereux C. - 71
Quilichini R. - 108
Rabineau D. - 87 - 91
Racadot E. - 127

Radal M. - 80 - 81
Raphael M. - 123
Réclar V. - 80 - 81
Réglier H. - 30
Renier G. - 44 - 124
Revenu M. - 7 - 16
Rigaut J.P. - 20
Robert-Nicoud M. - 40 - 43
Rocchi E. - 38
Roger P. - 45
Rohr M. - 115
Roignot P. - 63
Romagné F. - 114
Ronot X. - 27 - 32 - 37 - 40 - 43 - 100
Rossi J.F. - 132
Rotagnon M.O. - 97
Rougereau O. - 13
Roy L. - 86
Rummens J.L. - 109
Saadi A. - 62
Sabido O. - 70 - 94 - 96
Sainty D. - 114
Salaun V. - 106
Salmon J.M. - 38
Sampol J. - 26 - 31 - 54
Sanchez M. - 44
Sartiaux C. - 55 - 111
Schaaf-Lafontaine N. - 109
Schandelong A. - 126
Schiavon V. - 23
Schmitt D. - 63
Schoëvaërt D. - 20 - 48 - 49b
Schüpp S. - 8 - 10
Schurra C. - 135
Scotté M. - 101 - 102
Seigneurin D. - 27 - 32 - 100
Sibille B. - 37
Signolle J.P. - 3 - 13 - 14
Simony J. - 80 - 81
Sire S. - 39
Sohm E. - 48
Sola B. - 11 - 12
Solary E. - 111
Somerjai R. L. - 50
Sorokine-Durm I. - 86
Soumelis V. - 107
Soussaline F. - 86
Spyratos F. - 51
Staali L. - 39
Stoltz J.F. - 4
Talabard J.N. - 96
Tchernia G. - 84
Tenière P. - 101
Tertian G. - 84
Teyssier F. - 96
Thiry V. - 109
Tilly H. - 122
Touraine-Moulin F. - 97
Tracqui P. - 43
Triboulet J.P. - 73
Tron F. - 34 - 119 - 122
Trousard X. - 106
Truong K. - 83
Ts Li - 92
Vago P. - 51 - 58 - 68 - 80 - 81 - 104
Vandekerkhove P. - 109
Vandenh aute B. - 73
Vanhaeverbeek M. - 128
Vendrell JP - 56
Vergne M. - 47
Viallet P. - 38
Vielh P. - 83
Vigo J. - 38
Visseaux-Coletto B. - 71
Voisin P. - 86
Wallet F. - 17 - 18
Wattez N. - 74
Weber P. - 21
Xu C. - 67
Yatouji S. - 103
Zassadowski F. - 130
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