Complex of the herpes simplex virus type 1 origin binding protein UL9 with DNA as a platform for the design of a new type of antiviral drugs.

The herpes simplex virus type 1 origin-binding protein, OBP, is a DNA helicase encoded by the UL9 gene. The protein binds in a sequence-specific manner to the viral origins of replication, two OriS sites and one OriL site. In order to search for efficient inhibitors of the OBP activity, we have obtained a recombinant origin-binding protein expressed in Escherichia coli cells. The UL9 gene has been amplified by PCR and inserted into a modified plasmid pET14 between NdeI and KpnI sites. The recombinant protein binds to Box I and Box II sequences and possesses helicase and ATPase activities. In the presence of ATP and viral protein ICP8 (single-strand DNA-binding protein), the initiator protein induces unwinding of the minimal OriS duplex (≈80 bp). The protein also binds to a single-stranded DNA (OriS*) containing a stable Box I-Box III hairpin and an unstable AT-rich hairpin at the 3'-end. In the present work, new minor groove binding ligands have been synthesized which are capable to inhibit the development of virus-induced cytopathic effect in cultured Vero cells. Studies on binding of these compounds to DNA and synthetic oligonucleotides have been performed by fluorescence methods, gel mobility shift analysis and footprinting assays. Footprinting studies have revealed that Pt-bis-netropsin and related molecules exhibit preferences for binding to the AT-spacer in OriS. The drugs stabilize structure of the AT-rich region and inhibit the fluctuation opening of AT-base pairs which is a prerequisite to unwinding of DNA by OBP. Kinetics of ATP-dependent unwinding of OriS in the presence and absence of netropsin derivatives have been studied by measuring the efficiency of Forster resonance energy transfer (FRET) between fluorophores attached to 5'- and 3'- ends of an oligonucleotide in the minimal OriS duplex. The results are consistent with the suggestion that OBP is the DNA Holiday junction (HJ) binding helicase. The protein induces conformation changes (bending and partial melting) of OriS duplexes and stimulates HJ formation in the absence of ATP. The antiviral activity of bis-netropsins is coupled with their ability to inhibit the fluctuation opening of АТ base pairs in the А + Т cluster and their capacity to stabilize the structure of the АТ-rich hairpin in the single-stranded oligonucleotide corresponding to the upper chain in the minimal duplex OriS. The antiviral activities of bis-netropsins in cell culture and their therapeutic effects on HSV1-infected laboratory animals have been studied.

[Ultrasonic cleavage of DNA in complexes with Ag(I), Cu(II), Hg(II)].

We investigated a phenomenon of ultrasonic cleavage of DNA complexed with transition metal cations Ag(I), Cu(II) and Hg(II). We found the statistically significant dependence of relative intensity of cleavage on cation type and concentration. Each cation may cause two different types of distortion in the DNA double-helix depending on whether it binds to major or minor DNA groove. The intensity of ultrasonic cleavage decreases if cation binds to the major DNA groove; the intensity of cleavage increases if cation binds to the minor DNA groove and disturbs the hydrogen bonds of complementary base pairs or it intercalates between bases. Both types of DNA distortion can affect the intensity of N-S interconversion of deoxyribose.

[Inhibition of herpes simplex virus helicase UL9 by netropsin derivatives and antiviral activities of bis-netropsins].

Data obtained show that antiviral activities of bis-linked netropsin derivatives are targeted by specific complexes formed by helicase UL9 of herpes simplex virus type 1 with viral DNA replication origins, represented by two OriS sites and one OriL site. According to the results of footprinting studies bis-netropsins get bound selectively to an A+T-cluster which separates interaction sites I and II for helicase UL9 in OriS. Upon binding to DNA bis-netropsins stabilize a structure of the A+T-cluster and inhibit thermal fluctuation-induced opening of AT- base pairs which is needed for local unwinding of DNA by helicase UL9. Kinetics of ATP-dependent DNA unwinding in the presence and absence of Pt-bis-netropsin are studied by measuring the efficiency of Forster resonance energy transfer (FRET) between the fluorescent probes attached covalently to 3?- and 5?-ends of the oligonucleotides in the minimal OriS duplex. Pt-bis-netropsin and related molecules inhibit unwinding of OriS duplex by helicase UL9. Pt-bis-netropsin is also able to reduce the rate of unwinding of the AT- rich hairpin formed by the upper strand in the minimal OriS duplex. The antiviral activities and toxicity of bis-linked netropsin derivatives are studied in cell cultured experiments and experiments with animals infected by herpes virus.

[Analysis of DNA-ligand binding in solution and on biochips].

Ligand binding to DNA, as well as to microarrays, requires a system approach to description and analysis. This type of approach implies a fixed sequence of operations. Firstly, it is necessary to make a description of a binding scheme that realizes ligand and polymer in common spatial way. Secondly, a physical model of binding is required. Thirdly, a mathematical binding model should be constructed on the basis of the binding scheme and the physical model of binding. Every analysis of experimental data needs this preliminary work. A mathematical apparatus and classification of binding models have to follow on. Classification of different binding isotherms by different binding models is the direct problem. The inverse problem is a reconstruction of parameters of a binding model by experimental binding isotherm curves. The inverse problem can only be solved after solving the direct problem. An example of classification of binding models by oligonucleotides or proteins binding cooperativity and polymer properties like homo- or heteropolymer is presented.

Production of recombinant anthrax toxin receptor (ATR/CMG2) fused with human Fc in planta.

Mass vaccination against anthrax with existing vaccines is costly and unsafe due to potential side effects. For post-infection treatment, passive immunotherapy measures are currently available, most based on anthrax protective antigen (PA)-specific therapeutic antibodies. Efficient against wild-type strains, these treatment(s) might fail to protect against infections caused by genetically engineered Bacillus anthracis strains. A recent discovery revealed that the von Willebrand factor A (VWA) domain of human capillary morphogenesis protein 2 (CMG2) is an exceptionally effective anthrax toxin receptor (ATR) proficient in helping to resolve this issue. Here we describe in planta production of chimeric recombinant protein (immunoadhesin) comprised of functional ATR domain fused with the human immunoglobulin Fc fragment (pATR-Fc). The fusion design allowed us to obtain pATR-Fc in plant green tissues in a soluble form making it fairly easy to purify by Protein-A chromatography. Standardized pATR-Fc preparations (purity>90%) were shown to efficiently bind anthrax PA as demonstrated by ELISA and Western blot analysis. Recombinant pATR-Fc was also shown to protect J774A1 macrophage cells against the anthrax toxin. This study confirmed that plant-derived pATR-Fc antibody-like protein is a prospective candidate for anthrax immunotherapy.

[Properties of hybridization isotherms upon binding of ligands on microchips].

A simple thermodynamic model describing the microarray oligo-target hybridization has been constructed. The relationship between the hybridization signal intensity and Gibbs free energy for oligo-target duplex formation has been considered. The behavior of this function, which we called energetical hybridization isotherm, in response to target concentration change was modeled at different ratios of oligo-probes/target concentrations. The results of modeling were compared with the relevant and currently available data from microarray adsorption experiments.

[Statistical fluctuations of the filling level of operator by repressor determine the level of noise of reporter gene expression].

The regulation of the reporter gene activity in a single bacterial cell by means of lambda-phage C1 repressor has been described by the methods of statistical thermodynamics. The equations for the calculation of the mean production rate of the reporter protein and its standard deviation as a function of C1 repressor concentration in the cell have been obtained. The stochastic nature of C1 repressor binding with OR1 and OR2 operator sites becomes apparent when both repressor molecules and operators are present in the bacterial cell in a small number of copies. In this case, the number of repressor molecules that bind to OR1 and OR2 sites fluctuates considerably. The in vitro binding of C1 repressor to OR1 and OR2 sites, their mutant forms, and nonspecific DNA regions has been well studied. Using the binding constants of in vitro binding of C1 repressor to OR1, OR2 and nonspecific DNA regions and also the value of the cooperativity parameter for C1 repressor binding to OR1 and OR2 sites, we calculated the mean rate of synthesis of the reporter protein and its standard deviation as a function of repressor concentration in cell. The theoretical relations fit well the experimental results. The results of calculations confirm the assumption that gene expression noise in a single cell at a repressor concentration exceeding 100 nM is related to the stochastic nature of binding of repressor dimers to OR1 and OR2 sites. Other mechanisms of the generation of gene expression noise (for example, monomer-dimer balance) make a significant contribution at concentrations less than 100 nM.

Generation of plant-derived recombinant DTP subunit vaccine.

The current diphtheria-tetanus-pertussis (DTP) pediatric vaccine is produced from the corresponding pathogenic bacteria Corynebacterium diphtheriae, Clostridium tetani and Bordetella pertussis; five injected doses of DTaP (acellular) vaccine are required for every child in the standard US vaccination schedule. Because the vaccine is derived from native live sources, adverse effects are possible and production is complex and costly. To address issues of safety, ease of renewability and expense, we used recombinant technology in an effort to develop a subunit DPT vaccine derived in non-pathogenic plant expression systems. Expression of diphtheria toxin (DT), tetanus fragment-C (TetC) and the non-toxic S1 subunit of pertussis toxin (PTX S1) antigenic proteins in soluble form in low-alkaloid tobacco plants and carrot cell cultures allowed efficient downstream purification to levels suitable for intramuscular injection in BALB/c mice. At working concentrations of 5mug per dose, these preparations induced high levels of antigen-specific IgGs in mouse sera. Our results clearly support the feasibility of producing recombinant pediatric vaccine components in plants.

Immunological assessment of plant-derived avian flu H5/HA1 variants.

Polypeptide variants of the HA1 antigenic domain of the H5N1 avian influenza virus hemagglutinin (HA) molecule were produced in plants using transient and stable expression systems and fused with His/c-myc tags or with mouse or human Fc antibody fragments. The resulting peptides were purified and used for intramuscular immunization of mice. While the recombinant HA1 variants induced a significant serum humoral immune response in the mice, none of the HA1 preparations induced virus-neutralizing antibodies. Fusion with the Fc fragment improved overall yield of the constructs and allowed purification requiring only a single step, but led to no detectable fusion-related enhancement of immunogenicity or quality of immune response.

Advances in alfalfa mosaic virus-mediated expression of anthrax antigen in planta.

Plant viruses show great potential for production of pharmaceuticals in plants. Such viruses can harbor a small antigenic peptide(s) as a part of their coat proteins (CP) and elicit an antigen-specific immune response. Here, we report the high yield and consistency in production of recombinant alfalfa mosaic virus (AlMV) particles for specific presentation of the small loop 15 amino acid epitope from domain-4 of the Bacillus anthracis protective antigen (PA-D4s). The epitope was inserted immediately after the first 25 N-terminal amino acids of AlMV CP to retain genome activation and binding of CP to viral RNAs. Recombinant AlMV particles were efficiently produced in tobacco, easily purified for immunological analysis, and exhibited extended stability and systemic proliferation in planta. Intraperitional injections of mice with recombinant plant virus particles harboring the PA-D4s epitope elicited a distinct immune response. Western blotting and ELISA analysis showed that sera from immunized mice recognized both native PA antigen and the AlMV CP.

Interaction of a kinesin-like calmodulin-binding protein with a protein kinase.

Kinesin-like calmodulin-binding protein (KCBP) is a novel member of the kinesin superfamily that is involved in cell division and trichome morphogenesis. KCBP is unique among all known kinesins in having a myosin tail homology-4 region in the N-terminal tail and a calmodulin-binding region following the motor domain. Calcium, through calmodulin, has been shown to negatively regulate the interaction of KCBP with microtubules. Here we have used the yeast two-hybrid system to identify the proteins that interact with the tail region of KCBP. A protein kinase (KCBP-interacting protein kinase (KIPK)) was found to interact specifically with the tail region of KCBP. KIPK is related to a group of protein kinases specific to plants that has an additional sequence between subdomains VII and VIII of the conserved C-terminal catalytic domain and an extensive N-terminal region. The catalytic domain alone of KIPK interacted weakly with the N-terminal KCBP protein but strongly with full-length KCBP, whereas the noncatalytic region did not interact with either protein. The interaction of KCBP with KIPK was confirmed using coprecipitation assays. Using bacterially expressed full-length and truncated proteins, we have shown that the catalytic domain is capable of phosphorylating itself. The association of KIPK with KCBP suggests regulation of KCBP or KCBP-associated proteins by phosphorylation and/or that KCBP is involved in targeting KIPK to its proper cellular location.

A calmodulin binding protein from Arabidopsis is induced by ethylene and contains a DNA-binding motif.

Calmodulin (CaM), a key calcium sensor in all eukaryotes, regulates diverse cellular processes by interacting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35S-labeled CaM. A cDNA clone, EICBP (Ethylene-Induced CaM Binding Protein), encoding a protein that interacts with activated CaM was isolated in this screening. The CaM binding domain in EICBP was mapped to the C-terminus of the protein. These results indicate that calcium, through CaM, could regulate the activity of EICBP. The EICBP is expressed in different tissues and its expression in seedlings is induced by ethylene. The EICBP contains, in addition to a CaM binding domain, several features that are typical of transcription factors. These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. In database searches a partial cDNA (CG-1) encoding a DNA-binding motif from parsley and an ethylene up-regulated partial cDNA from tomato (ER66) showed significant similarity to EICBP. In addition, five hypothetical proteins in the Arabidopsis genome also showed a very high sequence similarity with EICBP, indicating that there are several EICBP-related proteins in Arabidopsis. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene.

An SC35-like protein and a novel serine/arginine-rich protein interact with Arabidopsis U1-70K protein.

The U1 small nuclear ribonucleoprotein 70-kDa protein, a U1 small nuclear ribonucleoprotein-specific protein, has been shown to have multiple roles in nuclear precursor mRNA processing in animals. By using the C-terminal arginine-rich region of Arabidopsis U1-70K protein in the yeast two-hybrid system, we have identified an SC35-like (SR33) and a novel plant serine/arginine-rich (SR) protein (SR45) that interact with the plant U1-70K. The SR33 and SR45 proteins share several features with SR proteins including modular domains typical of splicing factors in the SR family of proteins. However, both plant SR proteins are rich in proline, and SR45, unlike most animal SR proteins, has two distinct arginine/serine-rich domains separated by an RNA recognition motif. By using coprecipitation assays we confirmed the interaction of plant U1-70K with SR33 and SR45 proteins. Furthermore, in vivo and in vitro protein-protein interaction experiments have shown that SR33 protein interacts with itself and with SR45 protein but not with two other members (SRZ21 and SRZ22) of the SR family that are known to interact with the Arabidopsis full-length U-70K only. A Clk/Sty protein kinase (AFC-2) from Arabidopsis phosphorylated four SR proteins (SR33, SR45, SRZ21, and SRZ22). Coprecipitation studies have confirmed the interaction of SR proteins with AFC2 kinase, and the interaction between AFC2 and SR33 is modulated by the phosphorylation status of these proteins. These and our previous results suggest that the plant U1-70K interacts with at least four distinct members of the SR family including SR45 with its two arginine/serine-rich domains, and the interaction between the SR proteins and AFC2 is modulated by phosphorylation. The interaction of plant U1-70K with a novel set of proteins suggests the early stages of spliceosome assembly, and intron recognition in plants is likely to be different from animals.

The plant U1 small nuclear ribonucleoprotein particle 70K protein interacts with two novel serine/arginine-rich proteins.

The U1 small nuclear ribonucleoprotein particle (U1 snRNP) 70K protein (U1-70K), one of the three U1 snRNP-specific proteins, is implicated in basic and alternative splicing of nuclear pre-mRNAs. We have used the Arabidopsis U1-70K in the yeast two-hybrid system to isolate cDNAs encoding proteins that interact with it. This screening has resulted in the isolation of two novel plant serine/arginine-rich (SR) proteins, SRZ-22 and SRZ-21 (SRZ proteins). Neither the N-terminal region nor the arginine-rich C-terminal region of U1-70K alone interact with the SRZ proteins. The interaction of U1-70K with the SRZ proteins is confirmed further in vitro using a blot overlay assay. The plant SRZ proteins are highly similar to each other and contain conserved modular domains unique to different groups of splicing factors in the SR family of proteins. SRZ proteins are similar to human 9G8 splicing factor because they contain a zinc knuckle, precipitate with 65% ammonium sulfate, and cross-react with the 9G8 monoclonal antibody. However, unlike the 9G8 splicing factor, SRZ proteins contain a glycine hinge, a unique feature in other splicing factors (SC35 and ASF/SF2), located between the RNA binding domain and the zinc knuckle. SRZ-22 and SRZ-21 are encoded by two distinct genes and are expressed in all tissues tested with varied levels of expression. Our results suggest that the plant SRZ proteins represent a new group of SR proteins. The interaction of plant U1-70K with the SRZ proteins may account for some differences in pre-mRNA splicing between plants and animals.

Cloning of the cDNA for glutamyl-tRNA synthetase from Arabidopsis thaliana.

We cloned and characterized a full-length cDNA that encodes a glutamyl-tRNA synthetase (GluRSAt) from Arabidopsis. The GluRSAt is coded by a single gene. A transcript of about 2.3 kb hybridized with the cDNA. The deduced protein from the cDNA contained 719 amino acids with an estimated molecular mass of 81 kDa. Expression of the GluRSAt in E. coli resulted in a protein of the expected size. Comparison of the amino acid sequence GluRSAt to other glutamyl-tRNA synthetases showed strong sequence similarity to cytoplasmic GluRS proteins.

In vitro motility of AtKCBP, a calmodulin-binding kinesin protein of Arabidopsis.

AtKCBP is a calcium-dependent calmodulin-binding protein from Arabidopsis that contains a conserved kinesin microtubule motor domain. Calmodulin has been shown previously to bind to heavy chains of the unconventional myosins, where it is required for in vitro motility of brush border myosin I, but AtKCBP is the first kinesin-related heavy chain reported to be capable of binding specifically to calmodulin. Other kinesin proteins have been identified in Arabidopsis, but none of these binds to calmodulin, and none has been demonstrated to be a microtubule motor. We have tested bacterially expressed AtKCBP for the ability to bind microtubules to a glass surface and induce gliding of microtubules across the glass surface. We find that AtKCBP is a microtubule motor protein that moves on microtubules toward the minus ends, with the opposite polarity as kinesin. In the presence of calcium and calmodulin, AtKCBP no longer binds microtubules to the coverslip surface. This contrasts strikingly with the requirement of calmodulin for in vitro motility of brush border myosin I. Calmodulin could regulate AtKCBP binding to microtubules in the cell by inhibiting the binding of the motor to microtubules. The ability to bind to calmodulin provides an evolutionary link between the kinesin and myosin motor proteins, but our results indicate that the mechanisms of interaction and regulation of kinesin and myosin heavy chains by calmodulin are likely to differ significantly.

Structure and expression of a plant U1 snRNP 70K gene: alternative splicing of U1 snRNP 70K pre-mRNAs produces two different transcripts.

The product of the U1 small nuclear ribonucleoprotein particle (U1 snRNP) 70K (U1-70K) gene, a U1 snRNP-specific protein, has been implicated in basic as well as alternative splicing of pre-mRNAs in animals. Here, we report the isolation of full-length cDNAs and the corresponding genomic clone encoding a U1-70K protein from a plant system. The Arabidopsis U1-70K protein is encoded by a single gene, which is located on chromosome 3. Several lines of evidence indicate that two distinct transcripts (short and long) are produced from the same gene by alternative splicing of the U1-70K pre-mRNA. The alternative splicing involves inclusion or exclusion of a region (910 bp) that we named "included intron." Two transcripts were clearly detectable in all tissues tested, and the level of the transcripts varied in different organs. The deduced amino acid (427 residues) sequence from the short transcript has strong homology to the animal U1-70K protein and contains an RNA recognition motif, a glycine hinge, and an arginine-rich region characteristic of the animal U1-70K protein. The long transcript has an in-frame translational termination codon within the 910-bp included intron, resulting in a truncated protein containing only 204 amino acids. The protein encoded by the short transcript is recognized by U1 RNP-specific monoclonal antibodies and binds specifically to the Arabidopsis U1 snRNA, whereas the protein from the long transcript does not. In addition, multiple polyadenylation sites were observed in the 3' untranslated region. These results suggest a complex post-transcriptional regulation of Arabidopsis U1-70K gene expression.

A plant kinesin heavy chain-like protein is a calmodulin-binding protein.

Calmodulin, a calcium modulated protein, regulates the activity of several proteins that control cellular functions. A cDNA encoding a unique calmodulin-binding protein, PKCBP, was isolated from a potato expression library using protein-protein interaction based screening. The cDNA encoded protein bound to biotinylated calmodulin and 35S-labeled calmodulin in the presence of calcium and failed to bind in the presence of EGTA, a calcium chelator. The deduced amino acid sequence of the PKCBP has a domain of about 340 amino acids in the C-terminus that showed significant sequence similarity with the kinesin heavy chain motor domain and contained conserved ATP- and microtubule-binding sites present in the motor domain of all known kinesin heavy chains. Outside the motor domain, the PKCBP showed no sequence similarity with any of the known kinesins, but contained a globular domain in the N-terminus and a putative coiled-coil region in the middle. The calmodulin-binding region was mapped to a stretch of 64 amino acid residues in the C-terminus region of the protein. The gene is differentially expressed with the highest expression in apical buds. A homolog of PKCBP from Arabidopsis (AKCBP) showed identical structural organization indicating that kinesin heavy chains that bind to calmodulin are likely to exist in other plants. This paper presents evidence that the motor domain has microtubule stimulated ATPase activity and binds to microtubules in a nucleotide-dependent manner. The kinesin heavy chain-like calmodulin-binding protein is a new member of the kinesin superfamily as none of the known kinesin heavy chains contain a calmodulin-binding domain. The presence of a calmodulin-binding motif and a motor domain in a single polypeptide suggests regulation of kinesin heavy chain driven motor function(s) by calcium and calmodulin.

A novel plant calmodulin-binding protein with a kinesin heavy chain motor domain.

Calmodulin, a ubiquitous calcium-binding protein, regulates many diverse cellular functions by modulating the activity of the proteins that interact with it. Here, we report isolation of a cDNA encoding a novel kinesin-like calmodulin-binding protein (KCBP) from Arabidopsis using biotinylated calmodulin as a probe. Calcium-dependent binding of the cDNA-encoded protein to calmodulin is confirmed by 35S-labeled calmodulin. Sequence analysis of a full-length cDNA indicates that it codes for a protein of 1261 amino acids. The predicted amino acid sequence of the KCBP has a domain of about 340 amino acids in the COOH terminus that shows significant sequence similarity with the motor domain of kinesin heavy chains and kinesin-like proteins and contains ATP and microtubule binding sites typical of these proteins. Outside the motor domain, the KCBP has no sequence similarity with any of the known kinesins, but contains a globular domain in the NH2 terminus and a putative coiled-coil region in the middle. By analyzing the calmodulin binding activity of truncated proteins expressed in Escherichia coli, the calmodulin binding region is mapped to a stretch of about 50 amino acid residues in the COOH terminus region of the protein. Using a synthetic peptide, the calmodulin binding domain is further narrowed down to a 23-amino acid stretch. The synthetic peptide binds to calmodulin with high affinity in a calcium-dependent manner as judged by electrophoretic mobility shift assay of calmodulin-peptide complex. The KCBP is coded by a single gene and is highly expressed in developing flowers and suspension cultured cells. Although many kinesin heavy chains and kinesin-like proteins have been extensively characterized at the biochemical and molecular level in evolutionarily distant organisms, none of them is known to bind calmodulin. The plant kinesin-like protein with a calmodulin binding domain and a unique amino-terminal region is a new member of the kinesin superfamily. The presence of a calmodulin-binding motif in a kinesin heavy chain-like protein suggests a role for calcium and calmodulin in kinesin-driven motor function(s) in plants.

Isolation of a new mitotic-like cyclin from Arabidopsis: complementation of a yeast cyclin mutant with a plant cyclin.

Cyclins, a large family of proteins, are the regulatory subunits of cyclin-dependent protein kinase that are essential activators of cell cycle progression in eukaryotes. Here we report isolation of a new cyclin cDNA (cyclbAt) from Arabibopsis cDNA libraries using polymerase chain reaction amplified cyclin-box sequences as probes. The deduced amino acid sequence of the isolated cDNA showed the highest sequence similarity with mitotic cyclins. However, the nucleotide and predicted amino acid sequence of cyclbAt is different from five other mitotic-like cyclins that have recently been isolated from the same system, indicating that it is a new mitotic-like cyclin. These results, together with previous reports, suggest that there are at least six different mitotic-like cyclins in Arabidopsis. Expression of cyclbAt in yeast G1 cyclin-minus mutant (DL1) rescued the cyclin-minus phenotype, demonstrating, that plant mitotic-like cyclin can complement cyclin function in yeast. Analysis of expression of cyclbAt in different tissues by reverse transcription-polymerase chain reaction using primers corresponding to a unique region of the cDNA showed that cyclbAt is differentially expressed in different tissues with highest expression in flowers and no detectable expression in leaves.