M ATERIALS

Standard reagents A nalar grade standard reagents were supplied by BD H/M erck, Fisons and Sigma

Enzym es Restriction enzymes were supplied by Bethesda Research Laboratories (GIBCO-BRL) and New England Biolabs. M M uLV reverse transcriptase and Red Hot Taq polym erase w ere from Advanced Biotechnologies. All other enzymes were from Boehringer M annheim.

Prim ers The m ajority of primers w ere supplied by Oswel in solution. Some of the prim ers were purchased by Genosys or Pharm acia as lyophilised powder and w ere resuspended in 1ml of Ix TE. All prim ers were diluted providing a final concentration of 50pmoles/100p,l o f reaction.

E lectrophoresis reagents High melting point agarose was from Sigma. 40% (w/v) acrylamide, 2% (w/v) bisacrylam ide solution was from Severn Biotech. 19:1 Sequagel (manual sequencing) and Sequagel XR (autom atic sequencing) acrylamide w ere from N ational diagnostics. TEM ED was from BDH.

M iscellaneous N ick Sephadex G-50 columns, RN A se inhibitors, pd(N)ô random hexam ers and dNTPs were supplied by Pharmacia. Antibiotics w ere from Sigma. RNAzolB was from Biogenesis. Hybond N"^ m em brane filters w ere supplied by Am ersham Pharm acia Biotech. The same com pany also supplied [a-^^P]dCTP, [a-^^P]ddATP, [a-^¥ ]d d C T P , [a-^ ¥ ]d d G T P , [a-

term inator cycle sequencing kits, rediprime DNA labelling kit and shrimp alkaline phosphatase. [a-^^S]dATP was supplied by DuPont. The dye

term inator cycle sequencing ready reaction with Am pliTaq DNA polym erase, FS was supplied by Advanced Biosystems. 0.24-9.5Kb RN A and 1Kb DNA size ladders were supplied by GIBCO-BRL and lOObp D N A size ladder was supplied by Promega. Prom ega also supplied the W izard m iniprep DNA purification kit. QIAGEN supplied the QIAquick gel extraction, PGR

purification, mini plasm id purification and maxi plasm id/cosm id purification kits. m RNA QuickPrep M icro purification kit was from Pharm acia and Micro- FastTrack mRNA isolation kit from Invitrogen.

C om m only used solutions and buffers Glassware and solutions used in all experiments were sterilised either by autoclaving (15psi,

121°C for 20-25 minutes) or by filter sterilisation through a 0.22|Ltm pore size “A crodisk” filters (Gelman Sciences).

‘C hloroform ’: refers to a 24:1 (v/v) mixture of chloroform and isoam yl alcohol.

‘P henol’ : tefers to phenol equilibrated with TE, pH 7.5

‘Polyacrylam ide’ : refers to a 19:1 mixture o f acrylam ide and bisacrylam ide for m anual gels

D enaturing solution: 1.5M NaCl, 0.5M NaOH

N eutralising solution: 1.5M NaCl, 0.5M Tris, pH 7.5, Im M ED TA

D EPC-treated water: 0.1% Diethyl pyrocarbonate added to ddHaO, incubated for 2 hours o/n at 37°C and then autoclaved

Sequencing plate’s bonding solution: 3ml 95% ethanol, 5 |il

y-m ethocyloxylpropyltrim ethoxy silane and 50ql of 10% acetic acid lOOx D enhart’s: 2% Eicoll, 2% polyvinylpyrrolidone, 2% BSA

lOx M OPS: 0.2M MOPS, 50mM sodium acetate, lOmM EDTA, pHV.O 20x SSC: 3M NaCl, 300mM sodium citrate, pH 7.0 with citric acid

lOx TBE: 890mM Tris-HCl, 890mM boric acid, 20M m EDTA Ix TE: lOmM Tris-HCl and Im M EDTA, pH 7.5

Loading buffer: 30% glycerol in TE plus brom ophenol blue

T K M l: 0.24gr Tris-HCl, 0.41gr MgClz, 0.15gr KCl, O.lSgr EDTA, 200m l dH 20

TKM 2: 0.24g Tris-HCl, 0.41g MgClz, 0.15g KCl, 0.15g EDTA, 4.68g NaCl, 200m l dHzO

M icrobiology m edia and bacterial strains

Tryptone, Yeast extract and Bacto agar were from Difco. L-broth (per litre): lOg Tryptone, 5g Yeast extract, 5g NaCl L-agar (per litre): as L-Broth plus 14g Bacto agar

2x TY broth (per litre): lOg Tryptone, lOg Yeast extract, 5g NaCl 2x TY agar (per litre): as 2x TY Broth plus 14g Bacto agar

D H 5 a com petent cells (host strain for pUC19, GIBCO-BRL)

Ampicillin: lOOmg/ml in ddH20, filter sterilised through a 0.2|xm filter. Stored at 4°C for a short term period used at a working concentration of 100p.g/ml Kanamycin: 25mg/ml in ddH20, filter sterilised through a 0.2pm filter. Stored at 4°C for a short term period used at a working concentration of 25pg/m l

DNA sam ples Control samples included the Centre d ’Etudes de Polym orphism e Hum aine (CEPH) families and a small panel of m ale and fem ale DNA samples from individuals in the M RC HBGU.

DN A from the mouse cell hybrids 3E7 (chr Y), HORL9X (chr X), HORLI (chr 15) ham ster cell hybrid 853 (chr Y) and their parents IR3E (mouse) and

W g l l (hamster) were kindly provided by Prof. S. Povey (M RC HBGU ,UCL).

DNA from YACs containing inserts that form a contig along the Y chrom osom e were kindly provided by N. Affara.

H um an and m ouse tissues The healthy adult hum an testis tissue was dissected from a testicular cancer patient. Hum an foetal tissues were obtained from the M RC embryonic tissue bank (Ham m ersm ith Hospital) and were provided by Dr. L. W ong. The sex of the tissues was not determ ined and their m enstrual age was determ ined by either hand or foot measurements.

M ouse testis and kidney were obtained from a m ale adult mouse, supplied by UCL biological services.

The hum an Y chrom osom e/testis specific cDNA selection library

The selection procedure was carried out by Dr. M ichael Lovett and Dr. Richard Del M astro using first-strand cDNA prepared by J. Cam eron from adult hum an testis RN A and 480 cosmids from each of the two Y

chrom osom e cosmid libraries (n=960). 4,608 cDNAs were selected and am plified in the plasm id pA M P 10. The clones w ere arrayed in 48 96-well m icrotitre plates in lOOpl of 2x TY agar m edium containing 100p.g/ml ampicillin.

Y -cosm ids used for the construction o f the cDNA selection library

The Y chrom osom e cosmids used for the construction of this cDNA selection library originated from two separate genomic libraries. The first was constructed at the Biom edical Sciences Division, Law rence Liverm ore

N ational Laboratory, CA 94550 U .S.A (L L 0 Y N C 0 3 ‘M ’) under the auspices o f the National Laboratory Gene Library project sponsored by the U S

D epartm ent of Energy. The library was donated to our group by Dr. P.J De

Jong. This library was prepared by flow sorting Y chrom osom es from the somatic cell hybrid J640-51 and then partially digesting these with the enzym e

Mbol. The digests were size fractionated and ligated into the cosm id vector Law rist 16. The library comprises -13 ,0 0 0 clones of which 82% are hum an,

13% are ham ster and 5% are non-recombinant.

The second library was prepared by Taylor et al., (1996). For this library, D N A from the somatic cell hybrid 3E7 was digested with the enzym e

S a u 3 h \ , size fractionated and ligated into the cosm id vector Lorist B. This library com prises 1728 clones and is thought to contain <10% contam ination with hum an chrom osom es 1 and 12, caused by the presence of small

fragm ents o f these two chrom osom es in the genom e of 3E7.