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Protein kinase C activation is an early event in mouse fertilization
Cell Biology International
Reports, Vol. 74, Abstracts Supplement
7990
w105
THE INTERACTION OF HUMAN SPERM NUCLEAR PROTEINS WITH ZINC Jean Rousseaux, Fabien Bianchi, Roselyne RousseauxPrevost, Pierre Sautiere. URA CNRS 409, IRCL, 59045 Lille, FRANCE. Nuclear condensation of human sperm is related to the presence of basic proteins, protamines.Two classes of protamines are present in the nucleus : Pl protamine (HPl, rich in cystein), P2 protamines (HPZ, HP3, HP4, rich in histidine and cystein). Most of the cystein are disulfide bridged, but protein - protein as well as protein - DNA interaction are still unknown. A role of zinc in stabilization of human sperm chromatin has been evoked by different works. The purpose of the present study was to determine the mechanisms of interaction between human protamines and zinc. Zinc chelate affinity chromatography of sperm nuclear proteins, clearly separated the two classes of human protamines : HP1 (low affinity) and HP2, HP3, HP4 (high affinity), when a competitor ligand, imidazole, was used for elution. The results show that histidine residues play a critical role for interaction with zinc. Determination, now in progress, of the number of zinc atoms bound per protamine molecule will define whether the zinc-protein complex may contain metal binding domains analogous to "zinc finger" domains, and their possible involvement in proteinDNA interaction.
ABWANctMRCXX4B-Awra3BIcxs-~Alisan Moore, Linda penfold*, David%= Medical xolecular Biology &Harry-=*, unit, v of BiN,unimity college, Icdml and *Institute of zcQlcgy, zool~SocietyofIadlYn,Lmdal Inm%3rtoidentifypmtxhsinvol~in sperm-agg--,~-(RI)was ?zliHd against purified lxsmter zsrmmal nmbram5.ScreeninJofal~gtll~ W3t.iscDWlibrazywithR4resultedintW selh of a clew &-tad 134. 9% bfusiar protein ermded by 134 was ahmm to specifically block hmmn qmxmzanapellmidabhiinq~~mrthermblct melysis using labelled I.34 as a pmbe rrrvcrdled a single poly A+ RNA aFpmadaa;ely 2.3 kb; m aii Sped~~aseninrattzstisorliverRNA. sesuracing of the 165 k-p 134 clrmealed a high dqres (902) of hanology with the r&man pmbsae inhbitor Cgms anda significant butlauerlevelofIxxmlogytothemxrseti ratcm-@n-likegenes.~ofthe huKmcntAlibmyusingl34resultedinthe isolatim of a 2kb clone (261) which also bybddhd to a 2.3 kb hm?m tastis RNA. HOweVer in-to134, 261h@idimdto a 2.3 kb ISA species in rat testis an3 to a disthct2WixanscriptinratliverRt?A.No othwrattismles-expreesed~that lvkddbdtoeither~. suApanrteabya grantfXQBtheWell-TTUSt. wl(J7
WI08
ABPERMTAtLPRYfElNBlNDBAYC-E LooPoFmoBofMA-
ClaudioPksano,SilviaBonaooorsiandMaurkbGaffl.
Centru di Genetba Evok&nistba det CNR, Unhwsita’ di Roma “La fSqien&, Fbma. exhibit Primary spermalocyte nuctei of D. trwkqa& three giantYchmosom bopefmedbytfbek/-5,kl-3 and&-lferWyhSofs.Themsmctum3produce satditeDNAtrensaippswhbfldonot~tottle large amounts of pmteins. We cytoplasm-havesuggestedthahypothasisthattheYbopsprovfdaa structurat-krthemmpermlentanted accumulatbn of non Y-encoded proteins im&ed in spamlkgenesis. Our approach to identify the protebs associated w&h the loopsisthepmdudbnofpolydonel-~t sperm-spadfbproteins. Thus mice ware immunked with the 55 kD fractkw exdsed from an BDB-plyacrytamide gel bededwitftDttw@etestisproteins. Therasuubg antiserum, designated as Ti , @ec&aUy rewted Mh the k~3bopoffMmaryspetmatoqbsandwlththaspemf tails. Bidimensknel Weatam bbt an&y&s showad that the T1antisarummaotswithasingtespot differentfmmthe tub&s. Moreover, the Tl antigen pwed to be testis specifbbecauseitwaadetectadintastesandseminal veeoicks but not in cmcesses. Thwwepurlfktthe satkosyt-urea-insot~ fractbn of the axonemes known to contain a famify of sperm-specific proteins, the tektins, h~bgmoteaMweightsintherangeofthetubutins. Bbttbg experiments showed that a protein of this fraction reacts with the Tl ant&rum but not wfth the anti-tubulin entibod&s.
69
Reports, Vol. 74, Abstracts Supplement
7990
w105
THE INTERACTION OF HUMAN SPERM NUCLEAR PROTEINS WITH ZINC Jean Rousseaux, Fabien Bianchi, Roselyne RousseauxPrevost, Pierre Sautiere. URA CNRS 409, IRCL, 59045 Lille, FRANCE. Nuclear condensation of human sperm is related to the presence of basic proteins, protamines.Two classes of protamines are present in the nucleus : Pl protamine (HPl, rich in cystein), P2 protamines (HPZ, HP3, HP4, rich in histidine and cystein). Most of the cystein are disulfide bridged, but protein - protein as well as protein - DNA interaction are still unknown. A role of zinc in stabilization of human sperm chromatin has been evoked by different works. The purpose of the present study was to determine the mechanisms of interaction between human protamines and zinc. Zinc chelate affinity chromatography of sperm nuclear proteins, clearly separated the two classes of human protamines : HP1 (low affinity) and HP2, HP3, HP4 (high affinity), when a competitor ligand, imidazole, was used for elution. The results show that histidine residues play a critical role for interaction with zinc. Determination, now in progress, of the number of zinc atoms bound per protamine molecule will define whether the zinc-protein complex may contain metal binding domains analogous to "zinc finger" domains, and their possible involvement in proteinDNA interaction.
ABWANctMRCXX4B-Awra3BIcxs-~Alisan Moore, Linda penfold*, David%= Medical xolecular Biology &Harry-=*, unit, v of BiN,unimity college, Icdml and *Institute of zcQlcgy, zool~SocietyofIadlYn,Lmdal Inm%3rtoidentifypmtxhsinvol~in sperm-agg--,~-(RI)was ?zliHd against purified lxsmter zsrmmal nmbram5.ScreeninJofal~gtll~ W3t.iscDWlibrazywithR4resultedintW selh of a clew &-tad 134. 9% bfusiar protein ermded by 134 was ahmm to specifically block hmmn qmxmzanapellmidabhiinq~~mrthermblct melysis using labelled I.34 as a pmbe rrrvcrdled a single poly A+ RNA aFpmadaa;ely 2.3 kb; m aii Sped~~aseninrattzstisorliverRNA. sesuracing of the 165 k-p 134 clrmealed a high dqres (902) of hanology with the r&man pmbsae inhbitor Cgms anda significant butlauerlevelofIxxmlogytothemxrseti ratcm-@n-likegenes.~ofthe huKmcntAlibmyusingl34resultedinthe isolatim of a 2kb clone (261) which also bybddhd to a 2.3 kb hm?m tastis RNA. HOweVer in-to134, 261h@idimdto a 2.3 kb ISA species in rat testis an3 to a disthct2WixanscriptinratliverRt?A.No othwrattismles-expreesed~that lvkddbdtoeither~. suApanrteabya grantfXQBtheWell-TTUSt. wl(J7
WI08
ABPERMTAtLPRYfElNBlNDBAYC-E LooPoFmoBofMA-
ClaudioPksano,SilviaBonaooorsiandMaurkbGaffl.
Centru di Genetba Evok&nistba det CNR, Unhwsita’ di Roma “La fSqien&, Fbma. exhibit Primary spermalocyte nuctei of D. trwkqa& three giantYchmosom bopefmedbytfbek/-5,kl-3 and&-lferWyhSofs.Themsmctum3produce satditeDNAtrensaippswhbfldonot~tottle large amounts of pmteins. We cytoplasm-havesuggestedthahypothasisthattheYbopsprovfdaa structurat-krthemmpermlentanted accumulatbn of non Y-encoded proteins im&ed in spamlkgenesis. Our approach to identify the protebs associated w&h the loopsisthepmdudbnofpolydonel-~t sperm-spadfbproteins. Thus mice ware immunked with the 55 kD fractkw exdsed from an BDB-plyacrytamide gel bededwitftDttw@etestisproteins. Therasuubg antiserum, designated as Ti , @ec&aUy rewted Mh the k~3bopoffMmaryspetmatoqbsandwlththaspemf tails. Bidimensknel Weatam bbt an&y&s showad that the T1antisarummaotswithasingtespot differentfmmthe tub&s. Moreover, the Tl antigen pwed to be testis specifbbecauseitwaadetectadintastesandseminal veeoicks but not in cmcesses. Thwwepurlfktthe satkosyt-urea-insot~ fractbn of the axonemes known to contain a famify of sperm-specific proteins, the tektins, h~bgmoteaMweightsintherangeofthetubutins. Bbttbg experiments showed that a protein of this fraction reacts with the Tl ant&rum but not wfth the anti-tubulin entibod&s.
69