Class Of Zinc Finger Proteins Research Articles

Disruption of zinc (II) binding and dimeric protein structure of the XIAP-RING domain by copper (I) ions.

Modulation of metalloprotein structure and function via metal ion substitution may constitute a molecular basis for metal ion toxicity and/or metal-mediated functional control. The X-linked Inhibitor of Apoptosis Protein (XIAP) is a metalloprotein that requires zinc for proper structure and function. In addition to its role as a modulator of apoptosis, XIAP has been implicated in copper homeostasis. Given the similar coordination preferences of copper and zinc, investigation of XIAP structure and function upon interaction with copper is relevant. The Really Interesting New Gene (RING) domain of XIAP is representative of a class of zinc finger proteins that utilize a bi-nuclear zinc-binding motif to maintain proper structure and ubiquitin ligase function. Herein, we report the characterization of copper (I) binding to the Zn2-RING domain of XIAP. Electronic absorption studies that monitor copper-thiolate interactions demonstrate that the RING domain of XIAP binds 5-6 Cu(I) ions and that copper is thermodynamically preferred relative to zinc. Repetition of the experiments in the presence of the Zn(II)-specific dye Mag-Fura2 shows that Cu(I) addition results in Zn(II) ejection from the protein, even in the presence of glutathione. Loss of dimeric structure of the RING domain, which is a requirement for its ubiquitin ligase activity, upon copper substitution at the zinc-binding sites, was readily observed via size exclusion chromatography. These results provide a molecular basis for the modulation of RING function by copper and add to the growing body of literature that describe the impact of Cu(I) on zinc metalloprotein structure and function.

GmSAP5, a soybean A20/AN1 domain-containing stress-associated protein gene activated by GmAREB3, increases drought stress resistance in soybean by mediating ABA signaling

Drought stress impairs crop growth and productivity. Stress-associated proteins (SAPs), a class of zinc finger proteins containing the A20/AN1 domain, function in various stress responses in plants. However, little is known about the function of SAPs in drought-stress responses in soybean, an oil and protein crop. We report that a GmSAP5 protein confers drought tolerance by increasing sensitivity to abscisic acid (ABA) and reducing stomatal aperture. Overexpression and RNA interference of GmSAP5 in soybean hairy roots resulted in elevated resistance and sensitivity to drought stress, respectively. ABA and proline contents increased in GmSAP5-overexpressing plants under water-deficit conditions. Lower water loss rates and higher relative water contents were observed in GmSAP5-overexpressing plants, resulting in increased drought-stress resistance. A yeast one-hybrid assay and luciferase transient transcriptional activity assay showed that GmAREB3, an AREB/ABF transcription factor, could bind to the promoter of GmSAP5 and activate its expression. These results suggest that GmSAP5 acts downstream of GmAREB3 and improves drought-stress resistance by mediating ABA signaling.

Identification and Expression Analysis of Zinc Finger A20/AN1 Stress-Associated Genes SmSAP Responding to Abiotic Stress in Eggplant

Stress-associated proteins (SAP), a class of zinc-finger proteins, have been identified as novel stress regulatory proteins in stress responses. However, SAP genes in eggplant (SmSAP) have been little reported. It has important significance in identifying SAP members, understanding the molecular mechanisms underlying stress responses, and tolerance. We performed a comprehensive study of the A20/AN1 domains, motifs, gene structures, phylogenetic relationships, chromosomal locations, gene replications, collinearity, cis-acting elements, and expression pattern responses to various abiotic stresses. Twenty-one SAP genes were identified in eggplant (SmSAP) and were localized on 10 chromosomes. A phylogenetic analysis revealed that most of the SmSAP proteins showed a high homology with the tomato SAP members, and 21 members were divided into four groups based on the homology of the SAP members in eggplant, tomato, rice, and Arabidopsis. Further analysis revealed that SmSAP proteins contain the characteristic A20/AN1 domains, the A20 domain composed of motif 2 (ILCINNCGFFGSPATMNLCSKCYKDMJLK). Four pairs of tandem duplications were found in eggplant, and 10 SmSAP genes had collinearity with SAP genes from Arabidopsis, potato, or tomato, but only four SmSAP genes were collinear with SAP genes in the three species mentioned above. Moreover, the promoters of SmSAP genes were predicted to contain many cis-acting elements that respond to abiotic stress and hormones. A qRT-PCR analysis of the four selected SmSAP genes exhibited diverse expression levels in response to various environmental stresses. These results provided a comprehensive analysis of the SmSAP genes and lay a solid foundation for improving the understanding of the functional diversification of SAP genes under various environmental stresses in eggplant.

Identification and Expression Analysis of Stress-Associated Proteins (SAPs) Containing A20/AN1 Zinc Finger in Cucumber.

Stress-associated proteins (SAPs) are a class of zinc finger proteins that confer tolerance to a variety of abiotic and biotic stresses in diverse plant species. However, in cucumber (Cucumis sativus L.), very little is known about the roles of SAP gene family members in regulating plant growth, development, and stress responses. In this study, a total of 12 SAP genes (named as CsSAP1-CsSAP12) were identified in the cucumber genome, which were unevenly distributed on six chromosomes. Gene duplication analysis detected one tandem duplication and two segmental duplication events. Phylogenetic analysis of SAP proteins from cucumber and other plants suggested that they could be divided into seven groups (sub-families), and proteins in the same group generally had the same arrangement of AN1 (ZnF-AN1) and A20 (ZnF-A20) domains. Most of the CsSAP genes were intronless and harbored a number of stress- and hormone-responsive cis-elements in their promoter regions. Tissue expression analysis showed that the CsSAP genes had a broad spectrum of expression in different tissues, and some of them displayed remarkable alteration in expression during fruit development. RT-qPCR results indicated that all the selected CsSAP genes displayed transcriptional responses to cold, drought, and salt stresses. These results enable the first comprehensive description of the SAP gene family in cucumber and lay a solid foundation for future research on the biological functions of CsSAP genes.

Genome-wide survey and expression analysis of the stress-associated protein gene family in desert poplar, Populus euphratica

Stress-associated proteins (SAPs) are a novel class of zinc finger proteins that extensively participate in abiotic stress responses. To date, no overall analysis and expression profiling of SAP genes in woody plants have been reported. Populus euphratica is distributed in desert regions and is extraordinarily adaptable to abiotic stresses. Thus, it is regarded as a promising candidate for studying abiotic stress resistance mechanisms of woody plants. In this study, 18 non-redundant SAP genes were identified from the genome of P. euphratica using basic local alignment search tool algorithms and functional domain verification. Among these 18 PeuSAP genes, 15 were intronless. To investigate the evolutionary relationships of SAP genes in P. euphratica and other Salicaceae plants, phylogenetic analyses were performed. Subsequently, the expression profiles of the 18 PeuSAP genes were analyzed in different tissues and under various stresses (drought, salt, heat, cold, and abscisic acid (ABA) treatment) using quantitative real-time PCR. Tissue expression analysis indicated that PeuSAPs showed no tissue specificity. PeuSAPs were induced by multiple abiotic stresses, especially drought, salt, and heat stresses, perhaps because of abundant cis-acting heat shock elements and drought-inducible elements in the promoter regions of the PeuSAPs. Moreover, single nucleotide polymorphisms (SNPs) variant analysis revealed many synonymous and non-synonymous SNPs in PeuSAP genes, but the zinc finger structure was conserved during evolution. These results provide an overview of the SAP gene family in P. euphratica and a reference for further functional research on PeuSAP genes.

Yin Yang 1 is a multi-functional regulator of adipocyte differentiation in 3T3-L1 cells

Yin Yang 1 (YY1) is an ubiquitously distributed transcription factor that belongs to the GLI-Kruppel class of zinc finger proteins. The mechanism by which YY1 regulates adipocyte differentiation remains unclear. In this study, we investigated the functional role of YY1 during adipocyte differentiation. During the early stage, YY1 gene and protein expression was transiently downregulated upon the induction of differentiation, however, it was consistently induced during the later stage. YY1 overexpression decreased adipocyte differentiation and blocked cell differentiation at the preadipocyte stage, while YY1 knockdown by RNA interference increased adipocyte differentiation. YY1 physically interacted with PPARγ (Peroxisome proliferator-activated receptor gamma) and C/EBPβ (CCAAT/enhancer-binding protein beta) respectively in 3T3-L1 cells. Through its interaction with PPARγ, YY1 directly decreased PPARγ transcriptional activity. YY1 ectopic expression prevented C/EBPβ from binding to the PPARγ promoter, resulting in the downregulation of PPARγ transcriptional activity. These results indicate that YY1 repressed adipocyte differentiation by repressing the activity of adipogenic transcriptional factors in 3T3-L1 cells.

MiR-34a inhibits the migration and invasion of esophageal squamous cell carcinoma by targeting Yin Yang-1.

Esophageal squamous cell carcinoma (ESCC), one of the most common gastrointestinal tumors, is known for its high mortality rate. microRNAs (miRNAs) have been reported to play important regulatory roles in cancer metastasis and progression. miR-34a has been demonstrated to be associated with the development of and metastasis in certain types of cancer via various target genes, but its function and targets in ESCC are unknown. The aim of this study was to examine whether the expression of miR-34a was significantly decreased in ESCC tissues, compared with normal esophageal tissues using RT-PCR and western blot analysis. The results showed that miR-34a overexpression increased apoptosis and decreased clonogenic formation, but inhibited invasion and migration in ESCC cells by suppressing MMP-2 and -9 expression. Yin Yang-1 (YY1), a widely distributed transcription factor that belongs to the GLI-Kruppel class of zinc finger proteins, was found to be a direct target of miR-34a in ESCC cell lines. Rescue experiments indicated that the suppressive effect of miR-34a on invasion and migration was mediated by activating YY1 expression. Results of the present study showed that miR-34a is associated with ESCC migration and provides a potential therapeutic and diagnostic target for ESCC.

CPSF30, a Novel Non-Classical Zinc Finger Protein that Utilizes Iron & Zinc Coordination for RNA Recognition

Zinc finger (ZF) proteins are eukaryotic proteins that are involved in transcriptional and translational regulation. ZFs utilize cysteine and histidine residues to coordinate zinc ions to fold and function properly. There are fourteen different classes of zinc finger proteins, which are classified based upon the ligands involved in zinc coordination, the ZF domain folds, and the proteins’ biological targets. Cleavage and Polyadenylation Specificity Factor 30 (CPSF30) is a ‘non-classical’ ZF protein that contains multiple Cys3His zinc binding domains and is involved in pre-mRNA regulation. CPSF30 contains 5 Cys3His domains and a Cys2HisCys zinc knuckle domain. A construct of CPSF30 construct containing only CPSF30's five Cys3His domains, denoted as CPSF305FE was prepared. This protein is predicted to have five zinc ions; however, UV-visible and ICP-MS analysis of the isolated construct revealed that CPSF305FE contains iron in addition to zinc in a 1:4:1 Fe:Zn:CPSF305FE stoichiometetry. XAS analysis identified the iron site as a 2Fe-2S cluster. CPSF30 is predicted to recognize AU-rich RNA sequences. To test this hypothesis, RNA binding studies using both electrophoretic mobility shift assays (EMSA) and fluorescence anisotropy (FA) of CPSF305FE with α-synuclein pre-mRNA (which contains an AU-rich sequence) along with a series of mutated RNA sequences were performed. These studies revealed that CPSF30 binds to α-synuclein pre-RNA with nanomolar affinity and requires the AU-rich sequence for binding. The binding interaction was best fit to a cooperative binding model, suggesting that the CPF30/RNA binding event is cooperative.

The Rice B-Box Zinc Finger Gene Family: Genomic Identification, Characterization, Expression Profiling and Diurnal Analysis

BackgroundThe B-box (BBX) -containing proteins are a class of zinc finger proteins that contain one or two B-box domains and play important roles in plant growth and development. The Arabidopsis BBX gene family has recently been re-identified and renamed. However, there has not been a genome-wide survey of the rice BBX (OsBBX) gene family until now.Methodology/Principal FindingsIn this study, we identified 30 rice BBX genes through a comprehensive bioinformatics analysis. Each gene was assigned a uniform nomenclature. We described the chromosome localizations, gene structures, protein domains, phylogenetic relationship, whole life-cycle expression profile and diurnal expression patterns of the OsBBX family members. Based on the phylogeny and domain constitution, the OsBBX gene family was classified into five subfamilies. The gene duplication analysis revealed that only chromosomal segmental duplication contributed to the expansion of the OsBBX gene family. The expression profile of the OsBBX genes was analyzed by Affymetrix GeneChip microarrays throughout the entire life-cycle of rice cultivar Zhenshan 97 (ZS97). In addition, microarray analysis was performed to obtain the expression patterns of these genes under light/dark conditions and after three phytohormone treatments. This analysis revealed that the expression patterns of the OsBBX genes could be classified into eight groups. Eight genes were regulated under the light/dark treatments, and eleven genes showed differential expression under at least one phytohormone treatment. Moreover, we verified the diurnal expression of the OsBBX genes using the data obtained from the Diurnal Project and qPCR analysis, and the results indicated that many of these genes had a diurnal expression pattern.Conclusions/SignificanceThe combination of the genome-wide identification and the expression and diurnal analysis of the OsBBX gene family should facilitate additional functional studies of the OsBBX genes.

A tool for examining the role of the zinc finger myelin transcription factor 1 (Myt1) in neural development: Myt1 knock-in mice

The Myt1 family of transcription factors is unique among the many classes of zinc finger proteins in how the zinc-stabilized fingers contact the DNA helix. To examine the function of Myt1 in the developing nervous system, we generated mice in which Myt1 expression was replaced by an enhanced Green Fluorescent Protein fused to a Codon-improved Cre recombinase as a protein reporter. Myt1 knock-in mice die at birth, apparently due to improper innervation of their lungs. Elimination of Myt1 did not significantly affect the number or distribution of neural precursor cells that normally express Myt1 in the embryonic spinal cord. Nor was the general pattern of differentiated neurons altered in the embryonic spinal cord. The Myt1 knock-in mice should provide an important tool for identifying the in vivo targets of Myt1 action and unraveling the role of this structurally distinct zinc finger protein in neural development.

Functional characterization of iron-substituted neural zinc finger factor 1: metal and DNA binding

Neural zinc finger factor 1 (NZF-1) is a nonclassical zinc finger protein involved in neuronal development. NZF-1 contains multiple copies of a unique CCHHC zinc-binding domain that recognize a promoter element in the beta-retinoic acid receptor gene termed beta-retinoic acid receptor element (beta-RARE). Previous studies have established that a two-domain fragment of NZF-1 bound with zinc is sufficient for specific DNA binding. Proper functioning of the nervous system relies heavily on iron and misregulation of this highly redox active metal has serious consequences. Several classes of zinc finger proteins have been shown to bind other metal ions, including iron. To determine if ferrous iron can coordinate to the metal-binding sites of NZF-1 and assess the functional consequences of such coordination, a fragment of NZF-1 that contains two zinc-binding domains, NZF-1 double finger (NZF-1-DF), was prepared. UV-vis spectroscopy experiments demonstrated that Fe(II) is capable of binding to NZF-1-DF. Upon reconstitution with either Fe(II) or Zn(II), NZF-1-DF binds selectively and tightly (nanomolar affinity) to its target beta-RARE DNA sequence, whereas apo-NZF-1-DF does not bind to DNA and instead aggregates.

Transcriptome profiling of Saccharomyces cerevisiae mutants lacking C2H2 zinc finger proteins

BackgroundThe budding yeast Saccharomyces cerevisiae is a eukaryotic organism with extensive genetic redundancy. Large-scale gene deletion analysis has shown that over 80% of the ~6200 predicted genes are nonessential and that the functions of 30% of all ORFs remain unclassified, implying that yeast cells can tolerate deletion of a substantial number of individual genes. For example, a class of zinc finger proteins containing C2H2 zinc fingers in tandem arrays of two or three is predicted to be transcription factors; however, seven of the thirty-one predicted genes of this class are nonessential, and their functions are poorly understood. In this study we completed a transcriptomic profiling of three mutants lacking C2H2 zinc finger proteins, ypr013cΔ,ypr015cΔ and ypr013cΔypr015cΔ.ResultsGene expression patterns were remarkably different between wild type and the mutants. The results indicate altered expression of 79 genes in ypr013cΔ, 185 genes in ypr015cΔ and 426 genes in the double mutant when compared with that of the wild type strain. More than 80% of the alterations in the double mutants were not observed in either one of the single deletion mutants. Functional categorization based on Munich Information Center for Protein Sequences (MIPS) revealed up-regulation of genes related to transcription and down-regulation of genes involving cell rescue and defense, suggesting a decreased response to stress conditions. Genes related to cell cycle and DNA processing whose expression was affected by single or double deletions were also identified.ConclusionOur results suggest that microarray analysis can define the biological roles of zinc finger proteins with unknown functions and identify target genes that are regulated by these putative transcriptional factors. These findings also suggest that both YPR013C and YPR015C have biological processes in common, in addition to their own regulatory pathways.

A Fungal Family of Transcriptional Regulators: the Zinc Cluster Proteins

The trace element zinc is required for proper functioning of a large number of proteins, including various enzymes. However, most zinc-containing proteins are transcription factors capable of binding DNA and are named zinc finger proteins. They form one of the largest families of transcriptional regulators and are categorized into various classes according to zinc-binding motifs. This review focuses on one class of zinc finger proteins called zinc cluster (or binuclear) proteins. Members of this family are exclusively fungal and possess the well-conserved motif CysX(2)CysX(6)CysX(5-12)CysX(2)CysX(6-8)Cys. The cysteine residues bind to two zinc atoms, which coordinate folding of the domain involved in DNA recognition. The first- and best-studied zinc cluster protein is Gal4p, a transcriptional activator of genes involved in the catabolism of galactose in the budding yeast Saccharomyces cerevisiae. Since the discovery of Gal4p, many other zinc cluster proteins have been characterized; they function in a wide range of processes, including primary and secondary metabolism and meiosis. Other roles include regulation of genes involved in the stress response as well as pleiotropic drug resistance, as demonstrated in budding yeast and in human fungal pathogens. With the number of characterized zinc cluster proteins growing rapidly, it is becoming more and more apparent that they are important regulators of fungal physiology.

Identification of Two Novel Zinc Finger Modules and Nuclear Localization Signal in Rat Spermatidal Protein TP2 by Site-directed Mutagenesis

Spermatidal protein TP2, which appears transiently during stages 12-16 of mammalian spermiogenesis, is a DNA condensing zinc metalloprotein with a preference to GC-rich DNA. We have carried out a detailed site-directed mutagenesis analysis of rat spermatidal protein TP2 to delineate the amino acid residues involved in coordination with two atoms of zinc. Two zinc fingers modules have been identified involving 4 histidine and 4 cysteine residues, respectively. The modular structure of the two zinc fingers identified in TP2 define a new class of zinc finger proteins that do not fall into any of the known classes of zinc fingers. Transfection experiments with COS-7 cells using wild type and the two zinc finger pocket mutants have shown that TP2 preferentially localizes to nucleolus. The nuclear localization signal in TP2 was identified to be (87)GKVSKRKAV(95) present in the C-terminal third of TP2 as a part of an extended NoLS sequence.

The Ancient Source of a Distinct Gene Family Encoding Proteins Featuring RING and C3H Zinc-Finger Motifs with Abundant Expression in Developing Brain and Nervous System

Intronless genes can arise by germline retrotransposition of a cDNA originating as mRNA from an intron-containing source gene. Previously, we described several members of a family of intronless mammalian genes encoding a novel class of zinc-finger proteins, including one that shows imprinted expression and one that escapes X-inactivation. We report here the identification and characterization of the Makorin ring finger protein 1 gene (MKRN1), a highly transcribed, intron-containing source for this family of genes. Phylogenetic analyses clearly indicate that the MKRN1 gene is the ancestral founder of this gene family. We have identified MKRN1 orthologs from human, mouse, wallaby, chicken, fruitfly, and nematode, underscoring the age and conservation of this gene. The MKRN gene family encodes putative ribonucleoproteins with a distinctive array of zinc-finger motifs, including two to four C3H zinc-fingers, an unusual Cys/His arrangement that may represent a novel zinc-finger structure, and a highly conserved RING zinc-finger. To date, we have identified nine MKRN family loci distributed throughout the human genome. The human and mouse MKRN1 loci map to a conserved syntenic group near the T-cell receptor β cluster (TCRB) in chromosome 7q34–q35 and chromosome 6A, respectively. MKRN1 is widely transcribed in mammals, with high levels in murine embryonic nervous system and adult testis. The ancient origin of MKRN1, high degree of conservation, and expression pattern suggest important developmental and functional roles for this gene and its expressed family members.

JAZ Requires the Double-stranded RNA-binding Zinc Finger Motifs for Nuclear Localization

We have cloned and characterized a novel zinc finger protein, termed JAZ. JAZ contains four C(2)H(2)-type zinc finger motifs that are connected by long (28-38) amino acid linker sequences. JAZ is expressed in all tissues tested and localizes in the nucleus, primarily the nucleolus. JAZ preferentially binds to double-stranded (ds) RNA or RNA/DNA hybrids rather than DNA. Mutation of individual zinc finger motifs reveals that the zinc finger domains are not only essential for dsRNA binding but are also required for its nucleolar localization, which demonstrates a complex trafficking mechanism dependent on the nucleic acid-binding capability of the protein. Furthermore, forced expression of JAZ potently induces apoptosis in murine fibroblast cells. Thus, JAZ may belong to a class of zinc finger proteins that features dsRNA binding and may regulate cell growth via the unique dsRNA binding properties.

Lir-2, lir-1 and lin-26 encode a new class of zinc-finger proteins and are organized in two overlapping operons both in Caenorhabditis elegans and in Caenorhabditis briggsae.

lin-26, which encodes a unique Zn-finger protein, is required for differentiation of nonneuronal ectodermal cells in Caenorhabditis elegans. Here, we show that the two genes located immediately upstream of lin-26 encode LIN-26-like Zn-finger proteins; hence their names are lir-1 and lir-2 (lin-26 related). lir-2, lir-1, and lin-26 generate several isoforms by alternative splicing and/or trans-splicing at different positions. On the basis of their trans-splicing pattern, their intergenic distances, and their expression, we suggest that lir-2, lir-1, and lin-26 form two overlapping transcriptional operons. The first operon, which is expressed in virtually all cells, includes lir-2 and long lir-1 isoforms. The second operon, which is expressed in the nonneuronal ectoderm, includes short lir-1 isoforms, starting at exon 2 and lin-26. This unusual genomic organization has been conserved in C. briggsae, as shown by cloning the C. briggsae lir-2, lir-1, and lin-26 homologs. Particularly striking is the sequence conservation throughout the first lir-1 intron, which is very long in both species. Structural conservation is functionally meaningful as C. briggsae lin-26 is also expressed in the nonneuronal ectoderm and can complement a C. elegans lin-26 null mutation.

Myelin transcription factor 1 (Myt1) of the oligodendrocyte lineage, along with a closely related CCHC zinc finger, is expressed in developing neurons in the mammalian central nervous system.

The establishment and operation of the nervous system requires genetic regulation by a network of DNA-binding proteins, among which is the zinc finger superfamily of transcription factors. We have cloned and characterized a member of the unusual Cys-Cys-His-Cys (also referred to as Cys2HisCys, CCHC, or C2HC) class of zinc finger proteins in the developing nervous system. The novel gene, Myt1-like (Myt1l), is highly homologous to the original representative of this class, Myelin transcription factor 1 (Myt1) (Kim and Hudson, 1992). The MYT1 gene maps to human chromosome 20, while MYT1L maps to a region of human chromosome 2. Both zinc finger proteins are found in neurons at early stages of differentiation, with germinal zone cells displaying intense staining for MyT1. Unlike Myt1, Myt1l has not been detected in the glial lineage. Neurons that express Myt1l also express TuJ1, which marks neurons around the period of terminal mitosis. The Myt1l protein resides in distinct domains within the neuronal nucleus, analogous to the discrete pattern previously noted for Myt1 (Armstrong et al.: 14:303-321, 1995). The developmental expression and localization of these two multifingered CCHC proteins suggests that each may play a role in the development of neurons and oligodendroglia in the mammalian central nervous system.

Isolation by PCR of a cDNA clone from pea petals with similarity to petunia and wheat zinc finger proteins.

The C2H2 TFIIIA/Krüppel class of zinc finger proteins are an important group of regulatory nucleic acid binding factors and have been extensively studied in humans, Drosophila and yeast. We have employed 3' RACE PCR, using a highly degenerate oligonucleotide primer, for the facile isolation of a C2H2 zinc finger protein cDNA (Pszf1) from pea petals. The Pszf1 cDNA open reading frame potentially encodes a protein with two widely separated zinc fingers similar to zinc finger proteins from petunia and wheat. This class of two-fingered zinc finger proteins, possessing a wide and variable linker sequence, appears to be unique to plants. Three regions outside the zinc finger domains are also conserved between the members of the plant zinc finger protein family and one of these regions is a candidate nuclear localisation signal. The Pszf1 amino acid sequence is most similar to that of the petunia Epf1 protein, they possess an interfinger linker sequence of approximately the same length and they have a similar expression pattern with maximal transcript accumulation in mature petals, suggesting that Pszf1 may be the pea homologue of the petunia Epf1 zinc finger gene.

Cloning and characterization of two yeast genes encoding members of the CCCH class of zinc finger proteins: zinc finger-mediated impairment of cell growth

Members of the CCCH zinc finger (Zf) protein family have in common two or more repeats of a novel Zf motif consisting of Cys and His residues in the form Cx sCx 5Cx 3H [where x is a variable amino acid (aa)]. We used a degenerate polymerase chain reaction (PCR) strategy to clone members of this gene family from Saccharomyces cerevisiae. The deduced aa sequences encoded by these genes, designated CTH1 and CTH2, share 46% overall identity and 59% similarity, largely due to the two highly conserved Zf domains. We found readily detectable expression of a 1.4-kb mRNA encoding Cthlp. The 1.1-kb mRNA encoding Cth2p was barely detectable under normal growth conditions; however, disruption of CTHI resulted in at least a threefold increase in CTH2 mRNA accumulation. No change in phenotype was detected following disruption of CTH1 and CTH2, either singly or together. In contrast, overexpression of the CTH genes or one of the related mammalian genes, tris-tetraprolin ( TTP), caused delayed entry of cell cultures into exponential growth, and a decrease in final cell density. Removal of the Zf domain of Cthlp by truncation or deletion completely reversed this slow growth phenotype, indicating that it was mediated through this highly conserved structural motif