TIBS 17 - SEPTEMBER 1992
PROTEINSEQUENCE MOTIFS A novel zinc finger coiledcoil domain in a family of nuclear proteins A family of proteins was recently recognized that contains a new zinc finger motif called the RING finger, defined as CX2CX9_27CXHX2CX2CX6_I7CX2C , where X is any amino acid and the subscript indicates the number of residues (Refs 1, 2; R. Lovering et al., unpublished). A maternally expressed transcription factor, Xenopus nuclear factor 7 (xnf7) 3, also possesses the RING finger motif, but in addition contains a second, novel CH domain located 39 amino acids downstream from the RING finger4. The motif defines a new class of CH structures (CX2HX7CX7CX2CXsHX2H) that we refer to as the B box and has been previously identified in a number of other proteins 4. These include the proto-oncogenic protein RET 5, Rpt-1, a down regulator of the interleukin 2 receptor gene and the human immunodeficiency virus (HIV) LTR6, and a human RNA-binding autoantigen SS-A/Ro7. Recently, two associated B box motifs (herein referred to as B1 and B2) have been found in two other RING finger-containing proteins. These are PML, a putative transcription factor found fused to RARc¢in promyelocytic leukemia translocations TM and the human oncogenic protein T1812. An alignment of all the B box motifs (Fig. la) and subsequent statistical analysis shows that the B1 and B2 domains of PML and T18 are related to the B box motif and that all the B domains have probably evolved from a common ancestor. Furthermore, the B2 domains of PML and T18 are more closely related to the xnf7 B box than are the B1 domains, so are therefore probably functionally analogous to the B box motif found in other family members. However, the B1 and B2 domains appear to form a subgroup of the family, as highlighted by the presence of an extra potential cysteine metal ligand (underlined in Fig. la) and the replacements of Cys for His and His for Cys at some positions (Fig. la). Recently, synthetic peptides of both the RING finger and B box motifs have been shown to bind zinc preferentially (R. Lovering et al., unpublished; B. Reddy et aL, unpublished). Also, the RING finger motif binds to DNA in a zinc-dependent manner in vitro (R. Lovering et al., unpublished). In addition to the two highly conserved zinc finger RING and B box domains, all
344
(a) RPT- I
C
AQHG EKL RLFCRKDMMVICWLCERSQE
H
RGH
SS-A/Ro
C
AVHG ERL HLFCEKDGKALCWVCAQSRK
H
RDH
RET
C
EKHR EPL KLYCEEDQMPICVVCDRSRE
H
RGH
xnf7
C
SEHD ERL KLYCKDDGTLSCVICRDSLK
H
ASH
TI 8 B2
C
PFHKKEQL
H
P M L B2
CSNPNHRTPTLTSIYCRG~SKPLCCSCALLDSSHS
ELKC
P M L BI
C
TRCK ESA DFWCFE~EQLLCAKCFEA
HQW
FLKH
C
TSCEDNAEANGFCVE~VEWLCKTCIRA
HQRVKFTKDH
TI 8 BI
(b)
KLYCET~DKLTCRDC
QLLE
KEH
Zinc F i n g e r D o m a i n RING finger
B box
Coiled-coil
(5-8)
XNF7
]
RPT-1
]1 [
RET
]1
SS-A/RO PML
T18
(32)
27)
B2
(32)
(22)
B=
Figure 1 (a) Alignment of proteins that contain the B box motif. The residues in bold type are the putative zinc metal ion ligands. The alignment was obtained automatically using the program AMPS13 with a PAM matrix of 250. Scores were calculated according to the algorithm of Needleman and Wunsch14 and are based on pairwise comparisons of each sequence after 100 randomizations. These were used to assess the significance of the alignment. B1 and B2 refer to the two B box motifs found in T18 and PML. (b) Diagrammatic representation of the conserved zinc finger and coiled-coil domains in the B box-containing family. In PML and T18 there are two B box-like zinc finger domains B1 and B2. Based on the statistical analysis described in (a), B2 is shown aligned with the XNF7 B box. The shaded boxes represent the RING finger, the empty boxes represent the B boxes, and the lined boxes represent the coiled-coil domain. The symbol [ ] in the first four family members represents a gap. The spacing between the domains is shown in parentheses.
members of the B box-containing family possess a predicted coiled-coil domain immediately carboxy-terminal to the B box domains (Fig. lb). In PML and T18 the coiled-coil domain is adjacent to the third zinc finger (B2), and the spacing between the B box (or B2) zinc finger and the coiled-coil is highly conserved among all members of the RING finger-B box family (Fig. lb). More significantly, three of the six B box-containing genes (RET, PML and T18) have transformation potential when found as translocations in humans and mice. In the case of PML, recombination with the retinoic acid receptor is associated with acute promyelocytic leukemia 9 while RET possesses transforming potential when the RING-B box fingers and coiled-coil
domains are recombined with a tyrosine kinase domain 5. T18 is a transforming protein that results from the fusion between a protein of unknown function containing the zinc fingers and coiled-coil domains and the B-rafproto-oncogene 12. In all of these fusions the zinc finger and coiled-coil domains are retained. This suggests that the B box and coiled-coil motifs in this .family may play an important role in the transformation potential of these altered proteins 11. Thus, it will be of great interest to further characterize the functions of this novel domain and to identify more members of this family of nuclear proteins that probably have important functions in cellular growth and differentiation. © 1992, Elsevier Science Publishers, (UK)
TIBS 1 7 - S E P T E M B E R
1992
References 1 Freemont, P. S., Hanson, I. M. and Trowsdale, J. (1991) Ceil 64, 483-484 2 Haupt, Y. et al. (1991) Cell 65,753-763 3 Reddy, B., Kloc, M. and Etkin, L. D. (1991) Dev. Biol. 148, 107-116 4 Reddy, B. and Etkin, L. D. (1991) Nucleic Acids Res. 19, 6330 5 Takahashi, M., Inaguma, Y., Hiai, H. and Hirose, F. (1988) Mol. Cell. Biol. 8, 1853-1856 6 Patarca, R. eta/. (1988) Prec. Natl Acad. Sci. USA 85, 2733-2737 7 Chan, E. K. L., Hamel, J. C., Buyon, J. P. and
A neurofilament-specific sequence motif Neurofilaments are the major cytoplasmic components of large axons 1. As such, they should possess binding sites for a variety of other components of the neuron. The carboxyl termini of the neurofilament proteins NF-M and NF-H protrude from the sides of neurofilaments and seem to be ideally situated to mediate interactions between neurofilaments and other components of the neuron< However, few proteins are known to bind to neurofilaments in general, and most of these do not appear to bind to the carboxy-terminal tails. I have previously described two copies of a 15-amino-acid peptide at the extreme carboxyl terminus of NF-M (Ref. 3). The region that contains these repeats is by far the most conserved region of the known neurofilament tail extensions, and also contains a third conserved sequence, which aligns with the first seven amino acids of these full repeats (Table I). More recently, a novel neurofilament subunit, known as c¢-internexin4 or 66 kDa (Ref. 5) has been described. The small carboxyterminal extension of this protein contains one 15-amino-acid sequence which is clearly homologous to that of the NF-M repeats (Table I). FASTA searches of the PIR database with the c¢-internexin/ 66 kDa peptide finds the appropriate regions of c¢-internexin, followed by the most conserved of the NF-M repeats to be the best matches, while searches with the most conserved repeat of NF-M find the c¢-internexin peptide to be the secondbest non-NF-M match. Even though the number of subunits and immunological characteristics of fish neurofilaments are quite different from those of mammals 6, the sequence of an ~ NF-M-like protein from the electric ray Torpedo also contains a peptide in a homologous position at the extreme carboxyl terminus, which is clearly related to the mammalian/avian consensus described here 7. This 15-amino-acid segment therefore seems to identify an evolutionarily conserved neurofilament-specific motif, which is © 1992,ElsevierSciencePublishers, (UK)
Tan, E. M. (1991) J. Clin. Invest. 87, 68-76 8 Goddard, A. D., Borrow, J., Freemont, P. S. and Solomon, E. (1991) Science 254, 1371-1374 9 Kakizuka, A. eta/. (1991) Cell 66, 663-674 l O d e The, H. eta/. (1991) Cell 66,675-684 11 Kastner, P. et al. (1991) EMBO J. 11, 629-642 12 Miki, T. et al. (1991) Prec. Natl Acad. Sci. USA 88, 5176-5171 13 Barton, G. J. (1990) Methods Enzymol. 183, 403-428 14 Needleman, S. B. and Wunsch C. D. (1970) J. Mol. Biol. 48, 443-453
BRAMHAM A. REDDY AND LAURENCE D. ETKIN Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.
PAUL S. FREEMONT Protein Structure Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London, UK WC2A 3PX.
Table I. Sequencehomologybetween neurofilament proteins
presumably of some functional significance. Inspection of the tnbu]in- : binding motifs found in the microtubn]e-associated proteins ~ and MAP2 shows some superficial similarities but no strong sequence h o m o l o g y 8. Since m a n y neurons t h a t w e r e t h o u g h t to lack neurofilaments in
both the developing and mature nervous system have now been shown to contain c¢-internexin/ 66 kDa5'9, it may be that all higher vertebrate neurons express a neurofilament protein containing a version of this 15-amino-acid sequence.
Protein
Human NF-M~° Rat NF-M11 Mouse NF-M12 Chicken NF-M~3 Human NFM Rat NF-M Mouse NF-M Chicken NF-M Human NF-M Rat NF-M Mouse NF-M . Chicken NF-M Torpedo NF-M7 Rat c¢-internexin/66 kDa4
Residue numbers
Sequence
825-831 754-760 758-764 774-780 849-863 778-792 782-796 792-806 891-905 820-834 824-838 834-848 760-773 475-489
EEKGVVT EEKGVVT EEKGVVT
Consensus
References 1 Shaw, G. (1991) in Neuronal Cytoskeleton (Burgoyne, R. D., ed.), pp. 185-214, Alan Liss 2 Hisanaga, S-H. and Hirokawa, N. (1988) J. Mol. Biol. 202,297-305 3 Shaw, G. (1989) Biochem. Biophys. Res. Commun. 162, 294-299 4 Fliegner, K. H., Ching, G. Y. and Liem, R. K. (1990) EMBO J, 9, 749-755 5 Chui, F. C. et al. (1989) Neuron 2, 1435-1445 6 Lasek, R. J., Phillips, L., Katz, M. J. and Autilio-Gambetti, L. (1985) Ann. N. Y. Acad. Sci. 455, 462-478 7 Linial, M. and Scheller, R. H. (1990) J. Neurochem. 54; 762-770 8 Kaplan, M. P., Chin, S. S., Fliegner, K. H. and Liem, R. K. (1990) J. Neurosci. 10, 2735-2748 9 Goedert, M., Crowther, R. A. and Garner, C. C.
EEKGVVT EEKVVVTKTVSKITS DDKVWTKKVEKITS DDKVVVTKKVEKITS GEXVVVTKKAEKITS EEKLVSTKKVEKVTS EEKLVSTKKVEKVTS EEKLVSTKKVEKVTS EEKLVSTKKVEKVTS QETIVSTKTVEK-TS EETVVSTKKTEKSTI EEKVVVTKKVEKVTS
(1991) Trends Neuresci. 14, 193-199 10 Myers, M. W. et al. (1987) EMBO J. 6, 1617-1626 11 Napolitano, E. W., Chin, S. S., Colman, D. R. and Liem, R. K. (1987) J. Neuresci. 7,
2590-2599 12 Levy, E., Liem, R. K., D'Eustachio, P. and Cowan, N. J. (1987) Eur. J. Biochem. 166,
71-77 13 Zopf, D., Dineva, B., Betz, H. and Gundelfinger, E. D. (1990) Nucleic Acids Res. 18, 521-529
GERRY SHAW Department of Neuroscience, University of Florida College of Medicine, Box J-244, Gainesville, FL 32610-0244, USA.
Protein Sequence Motifs Following on from the popularity of our series of reviews on protein sequence motifs, we have introduced, as a regular feature, a column for brief reports of new motifs or sequence homologies that have been recognized in published sequences. Contributions to this column should be short (less than 500 words plus one figure) and will be subject to peer review. Preference will be given to reports of motifs or sequence homologies with profound biological significance. All sequences examined should have been published elsewhere in full and/or be freely available in the appropriate databases (e.g. Genbank, SWlSSPROT), but the particular motif or sequence alignment noted should not have been described before. Adequate statistical evaluation and, where appropriate, structural correlations should be given.
345
PROTEINSEQUENCE MOTIFS A novel zinc finger coiledcoil domain in a family of nuclear proteins A family of proteins was recently recognized that contains a new zinc finger motif called the RING finger, defined as CX2CX9_27CXHX2CX2CX6_I7CX2C , where X is any amino acid and the subscript indicates the number of residues (Refs 1, 2; R. Lovering et al., unpublished). A maternally expressed transcription factor, Xenopus nuclear factor 7 (xnf7) 3, also possesses the RING finger motif, but in addition contains a second, novel CH domain located 39 amino acids downstream from the RING finger4. The motif defines a new class of CH structures (CX2HX7CX7CX2CXsHX2H) that we refer to as the B box and has been previously identified in a number of other proteins 4. These include the proto-oncogenic protein RET 5, Rpt-1, a down regulator of the interleukin 2 receptor gene and the human immunodeficiency virus (HIV) LTR6, and a human RNA-binding autoantigen SS-A/Ro7. Recently, two associated B box motifs (herein referred to as B1 and B2) have been found in two other RING finger-containing proteins. These are PML, a putative transcription factor found fused to RARc¢in promyelocytic leukemia translocations TM and the human oncogenic protein T1812. An alignment of all the B box motifs (Fig. la) and subsequent statistical analysis shows that the B1 and B2 domains of PML and T18 are related to the B box motif and that all the B domains have probably evolved from a common ancestor. Furthermore, the B2 domains of PML and T18 are more closely related to the xnf7 B box than are the B1 domains, so are therefore probably functionally analogous to the B box motif found in other family members. However, the B1 and B2 domains appear to form a subgroup of the family, as highlighted by the presence of an extra potential cysteine metal ligand (underlined in Fig. la) and the replacements of Cys for His and His for Cys at some positions (Fig. la). Recently, synthetic peptides of both the RING finger and B box motifs have been shown to bind zinc preferentially (R. Lovering et al., unpublished; B. Reddy et aL, unpublished). Also, the RING finger motif binds to DNA in a zinc-dependent manner in vitro (R. Lovering et al., unpublished). In addition to the two highly conserved zinc finger RING and B box domains, all
344
(a) RPT- I
C
AQHG EKL RLFCRKDMMVICWLCERSQE
H
RGH
SS-A/Ro
C
AVHG ERL HLFCEKDGKALCWVCAQSRK
H
RDH
RET
C
EKHR EPL KLYCEEDQMPICVVCDRSRE
H
RGH
xnf7
C
SEHD ERL KLYCKDDGTLSCVICRDSLK
H
ASH
TI 8 B2
C
PFHKKEQL
H
P M L B2
CSNPNHRTPTLTSIYCRG~SKPLCCSCALLDSSHS
ELKC
P M L BI
C
TRCK ESA DFWCFE~EQLLCAKCFEA
HQW
FLKH
C
TSCEDNAEANGFCVE~VEWLCKTCIRA
HQRVKFTKDH
TI 8 BI
(b)
KLYCET~DKLTCRDC
QLLE
KEH
Zinc F i n g e r D o m a i n RING finger
B box
Coiled-coil
(5-8)
XNF7
]
RPT-1
]1 [
RET
]1
SS-A/RO PML
T18
(32)
27)
B2
(32)
(22)
B=
Figure 1 (a) Alignment of proteins that contain the B box motif. The residues in bold type are the putative zinc metal ion ligands. The alignment was obtained automatically using the program AMPS13 with a PAM matrix of 250. Scores were calculated according to the algorithm of Needleman and Wunsch14 and are based on pairwise comparisons of each sequence after 100 randomizations. These were used to assess the significance of the alignment. B1 and B2 refer to the two B box motifs found in T18 and PML. (b) Diagrammatic representation of the conserved zinc finger and coiled-coil domains in the B box-containing family. In PML and T18 there are two B box-like zinc finger domains B1 and B2. Based on the statistical analysis described in (a), B2 is shown aligned with the XNF7 B box. The shaded boxes represent the RING finger, the empty boxes represent the B boxes, and the lined boxes represent the coiled-coil domain. The symbol [ ] in the first four family members represents a gap. The spacing between the domains is shown in parentheses.
members of the B box-containing family possess a predicted coiled-coil domain immediately carboxy-terminal to the B box domains (Fig. lb). In PML and T18 the coiled-coil domain is adjacent to the third zinc finger (B2), and the spacing between the B box (or B2) zinc finger and the coiled-coil is highly conserved among all members of the RING finger-B box family (Fig. lb). More significantly, three of the six B box-containing genes (RET, PML and T18) have transformation potential when found as translocations in humans and mice. In the case of PML, recombination with the retinoic acid receptor is associated with acute promyelocytic leukemia 9 while RET possesses transforming potential when the RING-B box fingers and coiled-coil
domains are recombined with a tyrosine kinase domain 5. T18 is a transforming protein that results from the fusion between a protein of unknown function containing the zinc fingers and coiled-coil domains and the B-rafproto-oncogene 12. In all of these fusions the zinc finger and coiled-coil domains are retained. This suggests that the B box and coiled-coil motifs in this .family may play an important role in the transformation potential of these altered proteins 11. Thus, it will be of great interest to further characterize the functions of this novel domain and to identify more members of this family of nuclear proteins that probably have important functions in cellular growth and differentiation. © 1992, Elsevier Science Publishers, (UK)
TIBS 1 7 - S E P T E M B E R
1992
References 1 Freemont, P. S., Hanson, I. M. and Trowsdale, J. (1991) Ceil 64, 483-484 2 Haupt, Y. et al. (1991) Cell 65,753-763 3 Reddy, B., Kloc, M. and Etkin, L. D. (1991) Dev. Biol. 148, 107-116 4 Reddy, B. and Etkin, L. D. (1991) Nucleic Acids Res. 19, 6330 5 Takahashi, M., Inaguma, Y., Hiai, H. and Hirose, F. (1988) Mol. Cell. Biol. 8, 1853-1856 6 Patarca, R. eta/. (1988) Prec. Natl Acad. Sci. USA 85, 2733-2737 7 Chan, E. K. L., Hamel, J. C., Buyon, J. P. and
A neurofilament-specific sequence motif Neurofilaments are the major cytoplasmic components of large axons 1. As such, they should possess binding sites for a variety of other components of the neuron. The carboxyl termini of the neurofilament proteins NF-M and NF-H protrude from the sides of neurofilaments and seem to be ideally situated to mediate interactions between neurofilaments and other components of the neuron< However, few proteins are known to bind to neurofilaments in general, and most of these do not appear to bind to the carboxy-terminal tails. I have previously described two copies of a 15-amino-acid peptide at the extreme carboxyl terminus of NF-M (Ref. 3). The region that contains these repeats is by far the most conserved region of the known neurofilament tail extensions, and also contains a third conserved sequence, which aligns with the first seven amino acids of these full repeats (Table I). More recently, a novel neurofilament subunit, known as c¢-internexin4 or 66 kDa (Ref. 5) has been described. The small carboxyterminal extension of this protein contains one 15-amino-acid sequence which is clearly homologous to that of the NF-M repeats (Table I). FASTA searches of the PIR database with the c¢-internexin/ 66 kDa peptide finds the appropriate regions of c¢-internexin, followed by the most conserved of the NF-M repeats to be the best matches, while searches with the most conserved repeat of NF-M find the c¢-internexin peptide to be the secondbest non-NF-M match. Even though the number of subunits and immunological characteristics of fish neurofilaments are quite different from those of mammals 6, the sequence of an ~ NF-M-like protein from the electric ray Torpedo also contains a peptide in a homologous position at the extreme carboxyl terminus, which is clearly related to the mammalian/avian consensus described here 7. This 15-amino-acid segment therefore seems to identify an evolutionarily conserved neurofilament-specific motif, which is © 1992,ElsevierSciencePublishers, (UK)
Tan, E. M. (1991) J. Clin. Invest. 87, 68-76 8 Goddard, A. D., Borrow, J., Freemont, P. S. and Solomon, E. (1991) Science 254, 1371-1374 9 Kakizuka, A. eta/. (1991) Cell 66, 663-674 l O d e The, H. eta/. (1991) Cell 66,675-684 11 Kastner, P. et al. (1991) EMBO J. 11, 629-642 12 Miki, T. et al. (1991) Prec. Natl Acad. Sci. USA 88, 5176-5171 13 Barton, G. J. (1990) Methods Enzymol. 183, 403-428 14 Needleman, S. B. and Wunsch C. D. (1970) J. Mol. Biol. 48, 443-453
BRAMHAM A. REDDY AND LAURENCE D. ETKIN Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.
PAUL S. FREEMONT Protein Structure Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London, UK WC2A 3PX.
Table I. Sequencehomologybetween neurofilament proteins
presumably of some functional significance. Inspection of the tnbu]in- : binding motifs found in the microtubn]e-associated proteins ~ and MAP2 shows some superficial similarities but no strong sequence h o m o l o g y 8. Since m a n y neurons t h a t w e r e t h o u g h t to lack neurofilaments in
both the developing and mature nervous system have now been shown to contain c¢-internexin/ 66 kDa5'9, it may be that all higher vertebrate neurons express a neurofilament protein containing a version of this 15-amino-acid sequence.
Protein
Human NF-M~° Rat NF-M11 Mouse NF-M12 Chicken NF-M~3 Human NFM Rat NF-M Mouse NF-M Chicken NF-M Human NF-M Rat NF-M Mouse NF-M . Chicken NF-M Torpedo NF-M7 Rat c¢-internexin/66 kDa4
Residue numbers
Sequence
825-831 754-760 758-764 774-780 849-863 778-792 782-796 792-806 891-905 820-834 824-838 834-848 760-773 475-489
EEKGVVT EEKGVVT EEKGVVT
Consensus
References 1 Shaw, G. (1991) in Neuronal Cytoskeleton (Burgoyne, R. D., ed.), pp. 185-214, Alan Liss 2 Hisanaga, S-H. and Hirokawa, N. (1988) J. Mol. Biol. 202,297-305 3 Shaw, G. (1989) Biochem. Biophys. Res. Commun. 162, 294-299 4 Fliegner, K. H., Ching, G. Y. and Liem, R. K. (1990) EMBO J, 9, 749-755 5 Chui, F. C. et al. (1989) Neuron 2, 1435-1445 6 Lasek, R. J., Phillips, L., Katz, M. J. and Autilio-Gambetti, L. (1985) Ann. N. Y. Acad. Sci. 455, 462-478 7 Linial, M. and Scheller, R. H. (1990) J. Neurochem. 54; 762-770 8 Kaplan, M. P., Chin, S. S., Fliegner, K. H. and Liem, R. K. (1990) J. Neurosci. 10, 2735-2748 9 Goedert, M., Crowther, R. A. and Garner, C. C.
EEKGVVT EEKVVVTKTVSKITS DDKVWTKKVEKITS DDKVVVTKKVEKITS GEXVVVTKKAEKITS EEKLVSTKKVEKVTS EEKLVSTKKVEKVTS EEKLVSTKKVEKVTS EEKLVSTKKVEKVTS QETIVSTKTVEK-TS EETVVSTKKTEKSTI EEKVVVTKKVEKVTS
(1991) Trends Neuresci. 14, 193-199 10 Myers, M. W. et al. (1987) EMBO J. 6, 1617-1626 11 Napolitano, E. W., Chin, S. S., Colman, D. R. and Liem, R. K. (1987) J. Neuresci. 7,
2590-2599 12 Levy, E., Liem, R. K., D'Eustachio, P. and Cowan, N. J. (1987) Eur. J. Biochem. 166,
71-77 13 Zopf, D., Dineva, B., Betz, H. and Gundelfinger, E. D. (1990) Nucleic Acids Res. 18, 521-529
GERRY SHAW Department of Neuroscience, University of Florida College of Medicine, Box J-244, Gainesville, FL 32610-0244, USA.
Protein Sequence Motifs Following on from the popularity of our series of reviews on protein sequence motifs, we have introduced, as a regular feature, a column for brief reports of new motifs or sequence homologies that have been recognized in published sequences. Contributions to this column should be short (less than 500 words plus one figure) and will be subject to peer review. Preference will be given to reports of motifs or sequence homologies with profound biological significance. All sequences examined should have been published elsewhere in full and/or be freely available in the appropriate databases (e.g. Genbank, SWlSSPROT), but the particular motif or sequence alignment noted should not have been described before. Adequate statistical evaluation and, where appropriate, structural correlations should be given.
345
