indicating that the stop codon o f my putative ORF was incorrect. Smeared higher molecular weight bands were obtained in the reverse orientation o f the group II clone (lane 6), but fainter equivalents were also present in all other lanes, indicating that these were artefacts.
Discussion
The presence o f runs o f asparagine repeats in Dictyostelium proteins is not uncommon (see examples in figure 6.9 and Shaw et a l, 1989). In addition, many D ictyostelium proteins also contain runs o f glutamine repeats (present in the histidine kinase A and PIK3 sequences shown) or threonine repeats (present in the dokA protein sequence shown). These are all encoded by AAT or AAC trinucleotide repeats in different reading frames. It is unclear whether the repeats have any significance or are mere peculiarities o f D ictyostelium which have no functional effect. The presence o f two regions o f repeated sequence would, however, normally be expected to make the D N A susceptible to recombination.
I have shown that AAT repeats were present in the inserts o f an original amplified bacteriophage library and were not cloning artefacts introduced at a later stage o f library construction or screening. This was unlikely anyway, because the yeast screen had isolated several independent inserts o f different lengths encoding fragments o f the same cD N A which all contained the repeats (see Chapter Five). I also searched for the presence o f intron splice sites in the sequence, in case the insert that I had isolated was actually derived from
contaminating genomic D N A present in the RNA preparation used to synthesise the library cD N A . D ictyostelium intron splice sites have consensus sequences o f /G U AAGU at the 5' exondntron junction and A UA G / at the 3 'junction, where underlined nucleotides are absolutely conserved and the splice site is marked by '/' (Grant et a l , 1990). Additionally, the introns are on average 94% A+U rich (Kimmel and Firtel, 1983). Neither the nrcA or group II cD N A s contain sequences with strong homology to the consensus splice sites. There are a few weakly matched sites, but the D N A regions between these putative 5' and 3' splice sites do not have a sufficiently high A+T content for them to feasibly be introns.
The asparagine repeats o f the NrcA protein are restricted to regions outside o f the conserved cyclin box and destruction box motifs and are found only in regions o f the protein which are also poorly conserved between other cyclins. Thus, Dictyostelium may have a tendency to replace functionally insignificant residues with asparagine residues, to offer an
Histidine kinase a (Wang et a i , 1996) MELKTFKDLNDDIIGDTSPVINTGD. PNPLRTÛÔQQIiaQQQQaaQ<2QQQOQOC<SQüOQQQC<2QQ'SQHHIPQQLYQKOQOQCHSHSYGNHSFIHNVSPTSPSYDINNN NNNNNNNNNNNNNNNNNNNNSNNNNNNNNNNKinmiNNYYYSPIENSNISKSLEESVLNCFPHNFNLNSSNNNYLNNSSSLHNIN SVNSLSNNNNNQTNOÇPINNN NNNNNNNNNNNSNNSNNSNNNNNGNNNNNITDSPTKSKRHSTYETNIGSHORRKSIQSLIANSAIHSFSKLKNKPLSSSTPSTVNTCGAVNNNSNNNNNNNNNSTGS LGAIPMDRSFDGNINTITEESTGGNNSPRSNCGSNCGSNGGIPLSPRNLSSLNSGVNVSPRNIHLNNLNNNSSNLPPLSPRHINFHINVSNLNNNNNNNINPNNNPN NSNNSNNNVSPRNNNHNISPRGSNISPRSNNGGSTTISPRNISNNNNIINNINNNNILTPPRNSPRLENVNPTNSPRLLATSLNSTLPIVSSLTSSNNNN’SNNNTN PSINNNNGRNGHCIQTISEEILGNKPWYNNGNNNNNNNTNNSTTSNNNITTNNNNNNNNNINNNVLSTPRKRTKGNHSKTNSLQDFETSSMNGGDDSISGAGSGGS LRRRNKDDNDEtTOGNSNNTNSNNSITONNNNNNNSSNNNNNNSNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNYHNGATMMMSHNHQSIGMSSSP k k n n f k p f s r n c s l m g m g r r a w a i i l g l f i v g s s i s i l a t l v l r y s e e n s i a d d f a r v a r d r f t m l r i e f n n r l y i t q t l s l l l s v f p s t s e d;^f v p f s k l w s d n a e GLEGIMWAPRVSNLDRYTWEIEHSVKIREIVTNPNNSSDMRDVPAAAASDYYPILFSEPOSSNDHFKGYNIYSDMWRRPSLNKTRDTGEKVSVASPYINKLANVPKN SRSNVLLYIYQAVYTYGKVLSTVEDRRHEVIGFASCRFFISRMVSASLi'RLTEEDSLDLYVFDLDSTPIGELIYYRASNAGNDDGSSPTNIMNGKMLEDRSDMIYYN TMNVGGRNWMIALRPSRKFTNKHYTFYPYAIGGVCMLLSALVSFWFAVNTKHNIKLSATNEDLHKEIYNRKLAEKALAESQERLELAMEGSEDAVWDWKVNTGELHI SSRWFOILKAHDTSYQSRTLYEELKSSSTNNLNFKGDSKNGGSNNGTFNLFKNGKVDSSSP<,iSITNVNTTNGGGGGELRKSNSGYLYNDELFSPIILEEMVSSPNTH OLAIWNMKFLAELIHPDDKOKFISEIKKTITRETAIMEIECRMRKKYGGYLYIIMRGKWSNETSFKDNSLRMAGTLRDMTSRKDMQRLILEKEAAEEANKAKSAFV a t v s h e v r t p l s g v i g v s d l l l e t n l s e e q r d y v q t i q k s s q a l l t i i n d i l d y s k l e s r q l k m e t l p f s i i e t c q a v i h m l s v a a n d d v d i l l r v p p n v p r i i f g d
AM R M R Q V LLN R LSN A IK FTSR G H V LTD ISV D D SIPPTN TEEEIIH LC ITIED TG IG IPQ SLFD SIFEPFSvADNSTTRKYGGTGLGLSITKRLIEEVMGGTIQVSSI
v g q g s k f k c i i p f l l p n t s p s d l n l i s p s s l p k p f i n r s p k s t y s f t d k k n s v p s t p i p s g d i l i n k v c l l i c r d t v t e l v f k e c l e w l g m i v k;»v p r n v i d s i k n t
ILN N N N N NN N NN N NN N NN SNN SSSIISPSSLD Y SD EN EH LD LV LID LEILTEH LK IPSN V PIIFITPTK FNISK HN G ILN KW ITKSPN lrRV ELIRRPA ITD KLIPII SKCIKSQVQFTSGSSQLQSaOANLÜ<JQLLHgQLCNNGQTLNNNYNSGGIGGGGGGGGSNTMNGSS(3JLSNiniNFG : TPLSSGLVLLVHTGRTPPLFNNNGNSIIPPL ELAVDHHGNvQaQLYQQQQQV NNSSGNFQQFYQCr NNNSNNSFTPTLPNENSNNSIMNNSLNNNNTTPSNVTPTLFTSSPLDLQGRDTPVLQPPAYRKKALIVEDN ELNRKVLAaLFKKIDWTISFAENGREALKEITGERCFDIVFMDCOMPVLDGFQTTKIIRSKERENNWKRMNIVALSAGSSSSFVCDCLDSGMDSFMGKPITLATLKD ALAKWGGYNN
Histidine kinase, dokA (Schuster et a l, 1996)
MSSPHIELHSQRTLSPQPSSNNFELTGNKSCALSASLNGSIDDLNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNDGDKNDKKLLIITNAPTPPLTPPO PTK TQ QTLLELN,KFQEQQOOQÇiQO.QQLCKQNKôQOSOKQQEEPPSSOCEEPPLSQQQQEOEQEQEQEQEOEEOSKÇKIEGKGGGGEEEECEGGGGGEGEGEEQFKE GDEEETRVLKLIQSTNNW IYW YRRSSLOINEASPKLIC-TPSVDRTLCILDSLPCPIFQIGIIENTENGNITLTGFANERLTTLLSCSSNLLVDSLKSIPSPHYNTVP SLESCNSIPINNLAPGLLPNTDPTKILDPYISLYNKK-SLKERYSNNNNNTNTNNNNNNNNNNNNNNNNNNNNNNNDTTTTTTTTTTTTTTTTTTTTTTTTNESDNNN n n n n n n n n n n n n n n n n n n n n e e ik::n t k e g e e i l h e s t n s t g t g n s i s r p n s i l q c i l d s f h e q k t i t e k v l l s n f l i r q q y k s r a t i k p f p d g c i c i f e y i e n i n l NYQQPPTSLNDRINSTNKLTNEMELLSLSPDSNHSHVvKLHNHNHNHHNHNHNNTTiRASSTDSPFIHSVSANSLSSMSSNSSVTSACASLSSNSSSNSNNNSNNAS SSNCSSNANSNNTNSSSSNCCCCCNNNNNNNTSSTSSTISSASSTKSKSYLHOftHKNSKVYSTLLNSFENLSFLLKSTPIIIWRADHNGEMVFFKKSDEIPIDEKKV VGNNFQDFLQWVPQTYRTNFQEMFvNSLKCGKIFEFLFWFQTP; NYVQLFKIAGYPIFHIDGKDCIASKRYLIGWLGVTYNYLINDGAEISIKGSANLDSMYEKFKI lYEMPNIERTKLTNNGISSGGDITNANNNGGSSESSNKILEKKKHLISEAEKECFLKCKETYNILFKLSLLGVMFSTFKGIILDANDMLLgTIGYTRGDLENGKIDW MLLTPPEHFEISARALwELKAKRWC<^PIEKAYIHKSGKRVPVLITSAMIDGSTE;fCITFVFDLSRYRÇAEMAAIEATRLKTQFITNISHELRTPCHGIVGMSQLLLD SQLTNTC'RDNIDSIKRSTDSLISLINDILDFSKLEYGKVTLENESFELLPMIEEVLDSOATAANRKGIDLIFVMGRDYPVPPVIFGDRNSLKKVLLNLVGNAVKFTE TGFVLLEISTDYESGDglSLRFTVKDSGIGIPENKIEJIFVPFGvIDGSFSRKYGGSGLGLSFCKELVALM GGYIRVESGDQEGGKGTTFW FAIKVSISSPSYLPNS VPAAN,iFFYPEYRPSHYNNVIGNGDSPKNVLIIESN,:M VIM SI0SILLSM KCECISASKAITALDLLESSKSTEN,;IDIIVCSDKSTFVaQILDYVTTEKVILYGVD PNSKYNENSKVYSYLVTPITHSKLISSILLSKNLKSKNSFLTTTNNTNNTNNTNNIENKSSIDSPLSITSTSSSIITPILTSNNLDLNNNNNNNNNSILVSNNGGVD GGNNVPSTLTTIQQSCPKKYILVAEDNDINIKVW RgLEKLGYTAIVGINGLKALEIIGSFPICLILLDCOM PC’MDGFTCSTILRQIEPTGÇRIPIIAM TANDSKDR CFEVGMDDYLSKPVRVDRLQKTLSDWIKTDENGNPTNSYNFYPLSYSLVYNNFIDTvJLKKEKNDD
Protein tyrosine phosphatase, ptp3 (Cam per et a l, 1996)
M ISSSM SYRHSTNSVYTLNPHLNIPISTSTTIPPTSFYANNTPEMIOSQSENTNTNNINNSSSNINNNNNNTPDSMSMSTSLSSSPSVSFNHLDLNSINNKINNNTT TNNNOTJNNITONDDKFDTNALKLSOTMIIKNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNSNSNIEINVPSIQFDNEPAMEVDSVAPL NVPSNHTRTTLAMHNTKSLSTSNIGLLNILPNgwSSSSSSLSSTTTTTTTTSSSLLMPgSLFNNSTYNNHHNNNNSSNAGIVGGLNGSTSSLPT.'AQVQLOgMQQCM QQHQQHQYKKANLSSLSTWDNNLNNNPMNTSTSSPAQPNASPFSFSSSSLFSNSSLSNSGSGSASTTSTSTSSSNSMSSSPPPSLKTSFSQLDEDREKMRLEFEMI KKPEMASKKSHKHHORHYSHNDLDNRKHDEEKFFSALv PNNYGKNRYHDVLPNESTRVRLTPIESGDGDYINANYINGEVFNSYRYYIACgAPLPSTIKDFWRMVWE e r s s v i v c l t k l e e n g k k k a d v y y p e t s q a q e y g s f w i h l h k k v m f k d i g v s s l h l y k k g e e f p r e w l l h y t w p d c g a p p s s s h i r t l s v m v n t f k a r g s a k n t n
GPVIVHCSAGIGRSGTFISININMAKIERFGNDPSOMNISIKDSVLELRRORRGMVQTLDgY IFIFK V IN D V LTD M G IR SLSSPSK R R SC EM IK STPM PR LD ISIPP PLTFTPK D FC SSISPSTD M IA SLSIITvM TO TLK FPPQ O Q.D N PFSKSSIK ISPSPLNSTN ISIPKN ;:vF{JHPFQIQ PQLDLNLgOSQ QQ SSQ O LN DN PPLN M SSNS IKFPPVTSLSSCHLFEDSKNNDNNNKQQOOQQQQQvKNNOQCSGFSHFLNNNNNNDNNGSSGGGFNGSFLFNSNNSGSSSTNSECENNNKNNNNNSNNNNNNNNNKN SDNNGTKDKDENDSCESPRVTPIKCF
Phosphatidylinositol-4,5-diphosphate 3-kinase, PIK3 (Zhou et a l , 1995)
EQLEFTIKDLIDFKENYDKLESTEgFKÇWSNLIKNIKENSLNNSNIYLTIPTTC'NLINNNNNNNNNNNNNNNNNNNNNNNNNNNVIIPSASTENKEENDNNNSNNNN NINLSPDSSITKDINITENKITEIKTTETKETSTGTSPLEKSPSKGFIISPKKPEEENEIEGETINNIAITOYTQGPSM LTLM KKKLENIKKNNNNNNNNGNGNNNS NNNNSNSNNNNNGISPSSSPPSHLNGNNNNNNSNNTNSNNTTNATTNSVGFSITMTOSNSLSVSKRMNKFKSWTSSKPTSSSIGFASSPvNNGKPUlISGSSRFFTS RgDSKIDLLKSPSSSPPTv'SDIFNENNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNEELINNNNNNNNDENYKIEETEESLKELLEKEKLENEEREKILKERNEID NLKKKNHLSKGYFMRACNASNDDGLEEEDIPLvDEHWETNVIVLLPCRHHVKVPGSSSSSIDSIRCLAWASGKMOGHLNLEKDEKFFTLRWCNKDWFDCiDTPLGHL IQYNLNYNNPT KPTNIKLELVLEDELCKERLVDLQSLEINNGRPSIWKSHIDDVLSFNRKLRELAMLAKP'/SNVPAARLTPYPPPKTIPEFFVIRVHLFKNgTKSL RCANNHTAFSLMTILSEKLKNTTPFDPTQYRFLITGINQYVDPNVPLLSVEYIVEKIKRKGEIDLTMVELLSLGLIIQQ0QgQQQQ<J0QQQQ0Qi2IENIDDENILKL NNGILNVLSKIEKPIREKDNCISSLTVTENLgVRLLHAHEIFASKASEIIGTDSPSIQLFIEAAVYFGGELLATQSSKLVSFQDTWWNEWVNIPLAVSNIPNGARM CLGLNARYRGDIFNIGWVGHRLFDSKGILNTFAPFSLLLWPGKINPIGTCVDNLESKDOAIIIAFEFKDYWPKTIHYEDDLIELISKDENGNELPWTMEEMDRVE OIILODPLYSLNKEERLLIWKSRYFCHTKFgALSKLLC-SVEWTNYKgVGEAFOLLKIWPTLSAVDALELLDPKFADCVEIREYTVKCLDCMSDYELEIYLLCLVCAI KHDVFHNSVLSLFLIGRVWgNMOVLGHPFFWHLRADIDNQEVCERFRVLSSGFLRYAPTgLMESFKREITTLRILENLAKRVKEVPYEKRKQYVENNLREEQSFPTE LFVPFDPSIRILNIIPEKCKSMDSAKVPLWVTFKNADPFAPPLQMIAKTGDDLRQDILTLQLLRLMDHMWKSODLDLHMTIYRCIATGMGTGLIEWPNSETAARIQ AGAGGVSGAFKQTPIANWLKNHNQTENSYQKAVSKFTLSCAGYCVATYVLGIGDRHNDNIMVDIHGHLFHIDFGHFLGNFKTFAGFQREKAPFVLTPDFVYVIGGKD SPNFAFFVDICCKAFNIIRSNAHVFINMFELM LSTGIPELRSENDIVYLRDKFRLDLTDAEASEYFKKLIHESIGTLTTTINFAIHIM AHRKNLVSGNSAPKIGSAS SLNLNKNKPSSgSKLDLSRSDLSRSDSSRSDSSRLDLSRSDKKNNKDNKEKEKEKEKEKEKENNDNNDKDNNNNSNNDTEKENSIDK
NrcA
MNQTLSNENDEVKRSSSNKRESPFPQEDTNKNHKRVALYDVTHaQNIQPQPFNVNNNDCLVNNNYNNNNNNNNNNNYNNKNLMAKPIQSNKNNSIITASNIPSTFNN TATNNSNNNNNNNNNNNINNNNNNNINIISNNNNNNNNNNNNNNNNNNNNNNNNNNNKLKS . TVNGGIKTENLPSKNNNDNNSNSDDSNNSNKTNvTQODNSNNEIA PPTKPNNNNNNNNNNNNNNNNNNNNNNNNNNLTENENNELNNIKNNNNNNNNNNNNNNNNNNNNNNNNNNNNKENNSLEKTFHAHIHADFSHVPHIDIDEQFGACOI LCAEYAEEIFDNARKN WKT. PTDYM- NQSELKPGMRAILIDWIVDIGCELGVKNETIYLSINILDRYLSLOPVTRNEFOMIGACAFFIAAKYEEYKGACPQFIIQS AGEFFNVDQLLECECKMLKTLNFSLCTPTIKFFLGRYLIAVGDSDISHVAHLFGELSLLEVNLINYPPSVIAAACVYLACLVLQKgWTTTLTYHCRVEVNDIYFQKC VRFIYDKFQSNESETYSRTIKSKYAYVFDIFNKYSSNgSSSAPFCNKQOLIYS
Figure 6.9 Dictyostelium proteins which contain runs o f asparagine repeats
The polypeptide sequences of several Dictyostelium N-rich proteins, in addition to that of NrcA, are shown. Asparagine residues are highlighted in bold. Glutamine residues also form repeats in many Dictyostelium proteins and are shown here as faint residues.
Similarity trees and alignments o f the first 60 residues o f the cyclin box (the most conserved region) o f many cyclins, including NrcA, were shown in figures 1.1 and 1.2, Using this analysis, Dictyostelium cyclin B is closely related to plant B-type cyclins. By comparison, NrcA is not closely related to any particular group o f cyclins but is more similar to A, B and E-type cyclins than to D or F-types. The sequence analysis described earlier in this chapter though suggests that nrcA is a mitotic cyclin.
Both novel classes o f cD N A able to rescue the cyclin-deficient yeast are related to known cyclins, demonstrating that the approach o f yeast complementation to detect new
D ictyostelium cyclins can be successfully used, whereas other approaches had not previously
worked. nrcA was not detected by low stringency Southern blot analysis with a probe containing most o f the Dictyostelium cD N A despite its extensive hom ology with mitotic cyclins (Luo et a l , 1994). This may be because although cyclin hom ology is strong at the amino acid level in certain regions o f the protein, homology at the D N A level is extremely weak.
Neither cyclin was isolated by extensive attempts using PCR amplifications with degenerate oligonucleotide primers based on several highly conserved regions within the cyclin box. This was despite strongly conserved cyclin box residues being reasonably w ell retained in the NrcA protein, as shown in figure 1.2.
I had a limited amount o f time left available to perform experiments and decided to concentrate exclusively on the nrcA clone, a full-length cD N A encoding a protein with strong cyclin homology, rather than the group II clone which was only a partial clone with very limited hom ology to cyclins.