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Recent developments in genome-wide transcript monitoring have led to a rapid accumulation of data from gene expression studies. Such projects highlight the need for methods to predict the molecular basis of transcriptional coregulation. A microarray project identified the 420 yeast transcripts whose synthesis displays cell cycle-dependent periodicity. We present here a statistical technique we developed to identify the sequence elements that may be responsible for this cell cycle regulation. Because most gene regulatory sites contain a short string of highly conserved nucleotides, any such strings that are involved in gene regulation will occur frequently in the upstream regions of the genes that they regulate, and rarely in the upstream regions of other genes. Our strategy therefore utilizes statistical procedures to identify short oligomers, five or six nucleotides in length, that are over-represented in upstream regions of genes whose expression peaks at the same phase of the cell cycle. We report, with a high level of confidence, that 9 hexamers and 12 pentamers are over-represented in the upstream regions of genes whose expression peaks at the early G(1), late G(1), S, G(2), or M phase of the cell cycle. Some of these sequence elements show a preference for a particular orientation, and others, through a separate statistical test, for a particular position upstream of the ATG start codon. The finding that the majority of the statistically significant sequence elements are located in late G(1) upstream regions correlates with other experiments that identified the late G(1)/early S boundary as a vital cell cycle control point. Our results highlight the importance of MCB, an element implicated previously in late G(1)/early S gene regulation, as most of the late G(1) oligomers contain the MCB sequence or variations thereof. It is striking that most MCB-like sequences localize to a specific region upstream of the ATG start codon. Additional sequences that we have identified may be important for regulation at other phases of the cell cycle.

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Sequence-dependent DNA curvature is known to play an important role in initiation of transcription of many genes. We compared the distribution of predicted intrinsic curvature of Escherichia coli and human promoter sequences with the distribution of curvature of randomly selected coding and non-coding fragments from these organisms. Different methods of curvature calculation were found to yield mostly similar overall tendencies of DNA curvature in all groups of sequences. According to all methods of calculation, E. coli promoters were found to be more curved than coding sequences from the same genome and random sequences with the same nucleotide composition. By contrast, the average curvature of human promoter sequences was only marginally greater than the average curvature of human coding sequences. Non-coding intron sequences were found to be the most curved of the human sequences examined. Based on these observations, we hypothesize about the role of DNA curvature in promoter sequences.

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Progression through the eukaryotic cell cycle is known to be both regulated and accompanied by periodic fluctuation in the expression levels of numerous genes. We report here the genome-wide characterization of mRNA transcript levels during the cell cycle of the budding yeast S. cerevisiae. Cell cycle-dependent periodicity was found for 416 of the 6220 monitored transcripts. More than 25% of the 416 genes were found directly adjacent to other genes in the genome that displayed induction in the same cell cycle phase, suggesting a mechanism for local chromosomal organization in global mRNA regulation. More than 60% of the characterized genes that displayed mRNA fluctuation have already been implicated in cell cycle period-specific biological roles. Because more than 20% of human proteins display significant homology to yeast proteins, these results also link a range of human genes to cell cycle period-specific biological functions.

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The de novo protein albebetin has been engineered (J. Mol. Biol. 1992, 225, 927-931) to form a predesigned tertiary fold that has not yet been observed in natural proteins. Analysis of albebetin expressed in a cell-free system and in Escherichia coli revealed its compactness, relative stability, and the secondary structure close to the predesigned one. The blast-transforming biological activity of human interferon was grafted to albebetin by attachment of an eight amino acid interferon fragment to the N-terminus of albebetin next to its first methionine residue. The chimeric protein was expressed in a wheat germ cell-free translation system and tested for its structural properties, receptor binding, and biological activity. According to the tests, albebetin incorporating the active interferon fragment has a compact and relatively stable structure, and binds the murine thymocyte receptor effectively. It activates the blast transformation reaction of thymocyte cells even more efficiently than human interferon at low concentrations.

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The de novo protein albebetin has been designed recently to form a predetermined tertiary fold that has not yet been observed in natural proteins. An eight amino acid fragment (131-138) of human interferon alpha(2) carrying the blast-transforming activity of the protein was attached to the N-terminus of albebetin next to its initiatory methionine residue. The gene of chimeric protein was expressed in a wheat germ cell-free translation system and synthesized protein was tested for its compactness and stability. Its ability for receptor binding was also studied. We have shown that albebetin with attached octapeptide is practically as compact as natural proteins of corresponding molecular weight and possesses high stability toward the urea-induced unfolding. It binds murine thymocyte receptor at a high affinity and activates the thymocyte blast transformation efficiently at a concentration of 10(-11) M.

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An investigation of the functional topography of thermolysin was carried out using frequency analysis of its primary and tertiary structures. The statistical validity of this approach was estimated for the enzyme active site, the substrate-binding pocket, the inter-domain interface and calcium-binding sites' predictions. We showed that frequency analysis of primary structure could be employed to predict the localization of contiguous parts of the inter-domain interface. The same approach appears to be unsuitable to a search for conformation-dependent enzyme active sites and substrate-binding pockets. In contrast, frequency analysis of the spatial neighborhood is not effective for predicting the inter-domain interface as distinct from the active site, substrate-binding pocket and calcium-binding sites. These differences should be taken into account when investigating and understanding protein structure-function relationships.

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A theoretical investigation of the functional topography of thermolysin molecule was carried out using frequency analysis of its primary and tertiary structures. The statistical validity of predictions was estimated for the enzyme active site, substrate-binding pocket, interdomain interface, and calcium-binding sites. It was shown that frequency analysis of primary structure could be employed to predict the localization of contiguous parts of the interdomain interface. Primary structure analysis cannot be used to search for the conformation-dependent enzyme active site and substrate-binding pocket. On the contrary, frequency analysis of interresidues contacts is not so effective for prediction of the interdomain interface as compared with active site, substrate-binding pocket, and calcium-binding sites. The set of original algorithms proposed could be used in searching for functional sites in various proteins.

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Monoclonal and polyclonal anti-hepatitis A (HAV) antibodies were used to search for peptides mimicking the antigenic determinants of HAV. Synthetic peptides VP1 115-139, VP1 117-139, VP1 126-139, VP2 69-99, VP2 80-99, VP3 45-57, VP3 137-150, were shown to bind the anti-HAV antibodies in ELISA. Peptides VP1 115-139, VP1 117-139, VP2 69-99 were utilized to produce the antipeptide antibodies. Mice were immunized with the free peptides or with their conjugates with ovalbumin. Only the free VP2 69-99 caused formation of HAV binding antibodies.

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Computer search for probable T-epitopes of hepatitis A virus capsid proteins was performed using an integrated set of programs. Eight segments of the VP1, VP2, VP3 and VP4 proteins were chosen and synthesised. Five peptides previously examined as probable B-epitopes were used as well. All the peptides were tested for their ability to stimulate proliferation of lymph node T-cells primed with synthetic peptides. Almost all predicted T-epitopes affected the T-cell proliferation. None of the peptides had mitogenic activity. We demonstrated that regions 17-33 and 276-298 of VP1 are possible immunodominant promiscuous sites activating lymphocytes of all mouse haplotypes.

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Computer search for probable T-epitopes of the hepatitis A virus capsid proteins was performed using developed integrated set of programmes. The following peptides were chosen and synthesised by solid phase technique: 75-92 VP1, 115-139 VP1, 209-221 VP1, 69-99 VP2, 80-99 VP2, 45-57 VP3, 137-150 VP3 and 1-23 VP4. Peptides 1-17 VP1, 10-33 VP1, 11-25 VP1, 75-85 VP1 and 276-298 VP1 previously examined as probable B-epitopes were used as well. All the peptides were tested for their ability to stimulate proliferation of lymph node T-cells primed with synthetic peptides. Almost all the predicted T-epitopes did affected the T-cell proliferation. 10-33 VP1 and 276-298 VP1 stimulated lymph node proliferation of all tested mouse strains. 107-126 VP1 and 115-126 VP1 did not influence proliferation of lymphocytes of mice primed with these peptides but stimulated proliferation of T-cells of F1 (CBA x C57Bl6) mice primed with 115-139 VP1.

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Biologically active fragment 131-138 of human interferon alpha 2 carrying blast-transforming activity of the protein was attached to the N-terminus of the de novo protein albebetin with predetermined tertiary structure by means of genetic engineering. The chimeric protein was expressed in a wheat germ cell-free translation system and tested for compactness, stability and biological activity. According to the tests used albebetin with interferon fragment has a compact and relatively stable structure. It binds murine thymocyte receptor with high affinity and activates efficiently thymocyte blast transformation at a concentration of 10(-11) M.

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At present the growth hormone and prolactin receptors were cloned along with their variant forms from human, rat, mouse, rabbit, bovine and sheep tissues. The functional topography of receptors is practically unknown. Because of the high price and difficulty of protein's total mutagenesis, it is reasonable to carry on a theoretical analysis of structures of receptors to predict the most probable ligand-binding sites. We studied the primary structures of known prolactin and growth hormone receptors using theoretical methods proved to be powerful in earlier structure--activity relationship investigations. We analyzed the secondary structure, conservative positions, hydrophilicity profiles of the growth hormone and prolactin receptors, and used the original method based on information theory to predict the sites which are promising for mutagenesis or peptide synthesis as probable ligand-binding sites. Three segments corresponding to the main conservative, hydrophilic and rare sites were predicted to form the ligand-binding determinant.

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Peptide (P6) representing amino acids 62-75 of HAV capsid protein VP3 was synthesized. Pure P6, octamer form of P6, and P6 conjugated to KLH were injected into rabbits. The sera against these preparations reacted with denaturized VP3 in immunoblotting tests; however, only anti-P6-KLH serum was capable of binding the native HAV.

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A comparison of a new method of searching for functionally important sites in proteins and peptide hormones with a previously published method is presented. All methods are based on information theory that holds that rare oligomers contain much information and, thus, are likely to be important for specific functional activity. Twenty-five proteins and peptide hormones with known receptor-binding sites were studied; the new method showed good predictive ability. However, taking into account short-range sequential preferences in protein structures, the predictive ability of methods based on information theory, at least for the receptor-binding site predictions, was not greatly improved.

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To determine whether hypotheses about the complementarity of amino acids based on the genetic code reflect the amino acid contact preferences found in natural proteins, the average contact probabilities for hypothetical complementary amino acid pairs were compared with those for all possible remaining pairs of the corresponding subset. A statistically significant preference was found for contact between amino acids with codons which had the same central nucleotide. Conversely, the contact probabilities for amino acids with complementary codons either did not exceed, or exceeded only insignificantly, the value for the corresponding remainder subset. The data obtained do not support the hypothesis for the complementarity of peptides coded by complementary RNA strands.

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An essential part of structure-functional studies of proteins is the search for sites responsible for specific functional activity. The information theory can be of much help in such a search. According to this theory, rarely occurring oligomers contain more information and thus are more likely to take part in forming the active site. We used frequencies of occurrences of amino acids (mean and for each polypeptide) to search for clusters of highly informative amino acids by the moving window smoothing method. In 16 out of 19 peptide hormones such sequences were active sites known from literature.

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A simple method for searching amphipathic helices based on estimation of correlation between hydrophobicity distribution and periodic function is proposed. The method was examined in a series of proteins with known T-cell epitopes, which are mostly amphipathic helices. The predictive power of the method is discussed.

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