HAP2 Gene Research Articles

Plasmodium vivax HAP2 genetic diversity and population structure from worldwide clinical samples. A potential Transmission-Blocking malaria vaccine candidate

BackgroundMalaria infections due to Plasmodium vivax are increasingly becoming a significant public health problem and are directly impacting malaria elimination efforts in many countries. Recent functional studies have shown that a pre-fertilising antigen; gamete membrane fusion protein HAP2/Generative Cell specific 1 (GCS1) domain significantly reduces the formation of P. vivax oocysts in mosquitos, implying that it could be developed as a transmission-blocking vaccine. MethodsIn this study, the genetic diversity, natural selection, haplotype network analysis and population structure of PvHAP2/GCS1 full-length gene using worldwide samples from 16 countries are determined and the implications for its use as a potential vaccine candidate against P. vivax are discussed. ResultsThe study revealed low levels of nucleotide diversity (π ∼ 0.003, SNPs = 95) and purifying selection for the full-length PvHAP2 gene indicating functional constraints. Among the 95 SNPs (65 synonymous and 30 non-synonymous), 15 non-synonymous substitutions were localised to the C-terminal domain of the protein (674a.a − 862a.a) majority of these non-synonymous substitutions were from two countries of Central Africa i.e. Cameroon and Gabon. Phylogeographic haplotype network analysis indicated that a major haplotype (Hap_2, n = 34) is shared among countries from Asian countries and South American countries. Population structure analysis using a robust Bayesian algorithm indicated the existence of two sub-populations of PvHAP2/GCS1 among the worldwide samples. ConclusionThis study suggests that, due to low genetic diversity across 15 countries, and shared haplotype identified, the HAP2 gene can be a good target for vaccine development as a transmission-blocking vaccine with the potential to provide strain-transcending immunity. The C-terminal region from two countries from Central Africa (Cameroon and Gabon) identified high number of non-synonymous substitutions, which may indicate new adaption of parasites from these countries. However, more detailed field based studies with a larger sample size from these countries are needed.

MOLECULAR CHARACTERIZATION OF HAP3 GENE IN CASSAVA (Manihot esculenta Crantz)

Cassava (Manihot esculenta Crantz) is one of the food crops that has higher resistance to dry land, so it is possible to grow it in dry land. Cassava is used as an alternative food crop to maintain world food security, because the tuber has a high carbohydrate value, is easy to plant and is able to support food diversification. Cassava in Indonesia already has several nationally superior varieties such as Adira 1 and Malang 6. The first stage of cassava breeding is knowing the characteristics of genes in these plants, one of which is HAP3. The purpose of this study was to characterize the HAP3 gene in cassava (Manihot esculenta Crantz) using a molecular approach with genomics and bioinformatics. The methods used were DNA isolation of cassava leaves of Adira 1 and Malang 6 varieties, amplification by PCR, analysis of PCR results, and data sequencing. The results showed that the HAP3 gene in cassava varieties Malang 6 and Adira 1 had 100% homology with cultivar AM560-2 in nucleotide and amino acid sequences. HAP3 cassava varieties Malang 6 and Adira 1 have the closest relationship with Hevea brasiliensis plants with a homology value of 90.91% and Ricinus communis 83.33% based on amino acid sequences. The size of HAP3 on cassava varieties Adira 1 and Malang 6 was 546 bp.

Identification of a module of HAP transcription factors for seed development in rice

Heme activator protein (HAP) is a CCAAT box-binding factor family conserved in all higher eukaryotes. HAP transcription factors (TFs) consist of three subunits: HAP2, HAP3, and HAP5. Using recent rice genome information, we identified 11 HAP2 genes, 11 HAP3 genes, and 12 HAP5 genes. A HAP2 and five HAP5 genes were acknowledged more in this study than in the previous studies. Due to the functional association of HAP subunits, we carried out clustering analysis for expression patterns of all HAP members using anatomical meta-expression data of 983 rice Affymetrix arrays collected from the public database. Subsequently, we identified HAP members with endosperm and seed-preferred expression and focused on nine HAP family members associated with this expression pattern. We confirmed their expression patterns by RT-PCR analyses. The nine members were OsHAP2G, OsHAP2I, OsHAP3D, OsHAP3K, and five HAP5 genes (LOC_Os05g23910, LOC_Os10g11580, LOC_Os01g24460, LOC_Os01g01290, and LOC_Os01g39850). Promoter analyses using GUS activity again demonstrated that OsHAP3K is preferentially expressed in the aleurone layer of the seed and the LOC_Os05g23910 gene-encoding HAP5 showed endosperm-preferred expression. Network analysis using RiceNet suggests a functional gene network mediated by four rice HAP genes, further helping in the prediction of the biological function of OsHAP2G and OsHAP5 (LOC_Os10g11580). Our data provide new insight into HAP family-mediated regulatory pathways for endosperm or seed development in rice.

Hap2, a novel gene in Babesia bigemina is expressed in tick stages, and specific antibodies block zygote formation

BackgroundBovine babesiosis is a tick-borne disease caused by the protozoan parasites of the genus Babesia. In their host vector, Babesia spp. undergo sexual reproduction. Therefore, the development of sexual stages and the subsequent formation of the zygote are essential for the parasite to invade the intestinal cells of the vector tick and continue its life-cycle. HAP2/GCS1 is a protein identified in plants, protozoan parasites and other organisms that has an important role during membrane fusion in fertilization processes. The identification and characterization of HAP-2 protein in Babesia would be very significant to understand the biology of the parasite and to develop a transmission-blocking vaccine in the future.ResultsTo isolate and sequence the hap2 gene DNA from an infected bovine with Babesia bigemina was purified. The hap2 gene was amplified, cloned and sequenced. The sequences of hap2 from four geographically different strains showed high conservation at the amino acid level, including the typical structure with a signal peptide and the HAP2/GSC domain. Antisera anti-HAP2 against the conserved extracellular region of the HAP2 amino acid sequence were obtained from rabbits. The expression of hap2 in the host and vector tissues was analyzed by using semi-quantitative RT-PCR, and the protein was examined by western blot and immunofluorescence. Based on the RT-PCR and WB results, HAP2 is expressed in both, sexual stages induced in vitro, and in infected ticks as well. We did not detect any expression in asexual erythrocytic stages of B. bigemina, relevantly anti-HAP2 specific antibodies were able to block zygotes formation in vitro.ConclusionBabesia bigemina HAP2 is expressed only in tick-infecting stages, and specific antibodies block zygote formation. Further studies regarding the function of HAP2 during tick infection may provide new insights into the molecular mechanisms of sexual reproduction of the parasite.

The Babesia bovis hap2 gene is not required for blood stage replication, but expressed upon in vitro sexual stage induction.

Babesia bovis, is a tick borne apicomplexan parasite responsible for important cattle losses globally. Babesia parasites have a complex life cycle including asexual replication in the mammalian host and sexual reproduction in the tick vector. Novel control strategies aimed at limiting transmission of the parasite are needed, but transmission blocking vaccine candidates remain undefined. Expression of HAP2 has been recognized as critical for the fertilization of parasites in the Babesia-related Plasmodium, and is a leading candidate for a transmission blocking vaccine against malaria. Hereby we identified the B. bovis hap2 gene and demonstrated that it is widely conserved and differentially transcribed during development within the tick midgut, but not by blood stage parasites. The hap2 gene was disrupted by transfecting B. bovis with a plasmid containing the flanking regions of the hap2 gene and the GPF-BSD gene under the control of the ef-1α-B promoter. Comparison of in vitro growth between a hap2-KO B. bovis clonal line and its parental wild type strain showed that HAP2 is not required for the development of B. bovis in erythrocytes. However, xanthurenic acid-in vitro induction experiments of sexual stages of parasites recovered after tick transmission resulted in surface expression of HAP2 exclusively in sexual stage induced parasites. In addition, hap2-KO parasites were not able to develop such sexual stages as defined both by morphology and by expression of the B. bovis sexual marker genes 6-Cys A and B. Together, the data strongly suggests that tick midgut stage differential expression of hap2 is associated with the development of B. bovis sexual forms. Overall these studies are consistent with a role of HAP2 in tick stages of the parasite and suggest that HAP2 is a potential candidate for a transmission blocking vaccine against bovine babesiosis.

Analysis of the gamete-fusion genes in the haploid-inducing ZMS-P maize line

This article is devoted to the study of the double fertilization mechanism in plants, in particular of the maize gamete membrane fusion genes. We detected and analyzed for the first time gamete-fusion genes in the maize genome. Using the BLAST program, we searched for the hap2 gene (generative cell specific 1 (gcs1)) homologs from Arabidopsis in the maize genome. The ZM_BFb0162K03 maize transcript was found, which had 67% identity to the Athap2 gene and contained a conserved region similar to the Athap2 gene fragment. In mRNA samples from the haploid-inducing and control maize lines, an PCR was conducted by using primers specific to the ZM_BFb0162K03 sequence fragment. Sequences of the PCR products from a fragment (1467 bp) of the Zm_hap2 gene of the haploid-inducing and the control maize lines were identical and also were identical to the maize sequences from the GenBank (ZM_BFb0162K03). PCR products (656 bp region of Zm_hap2) for the ZM_BFb0162K03 (1925 bp) maize sequence were observed for the cDNA of pollen grains, ovary, leaves, and roots of the haploid-inducing and control maize lines. Using the Blastx program, we found significant homology of the maize translated proteins to the GEX2, TET11, and TET12 proteins, involved in Arabidopsis gamete-fusion contacts.

In silico characterization and evolutionary analyses of CCAAT binding proteins in the lycophyte plant Selaginella moellendorffii genome: A growing comparative genomics resource

NF-Y transcription factors encoded by HAP gene family, composed of three subunits (HAP2/NF-YA, HAP3/NF-YB and HAP5/NF-YC), are capable of transcriptional regulation of target genes with high specificity by binding to the CCAAT-containing promoter sequences. Here, we have characterized duplicated HAP genes in Selaginella moellendorffii and explored some features that might be involved in the regulation of gene expression and their function. Subsequently, the evolutionary relationships of LEC1-type of HAP3 genes have been studied starting from lycophytes to angiosperm to reveal the details of conservation and diversification of these genes during plant evolution. Computational analyses demonstrated the variation in length of cis-regulatory region of HAP3 duplicates in S. moellendorffii containing three thermodynamically stable and evolutionarily conserved RNA secondary structures. The homology modeling of NF-Y proteins, secondary structural details, DNA binding large positive patches, binding affinity of H2A–H2B interactive residues of NF-YC subunits on the duplicated NF-YB subunits, conserved domain analyses and protein structural alignments indicated that gene duplication process of HAP genes in S. moellendorffii, followed by structural diversification, provide specific hints about their functional specificity under various circumstances for the survival of this lycophytic plant. We have identified several conserved motifs in LEC1 proteins among all plant lineages during evolution.

Embryogenic potential and expression of embryogenesis-related genes in conifers are affected by treatment with a histone deacetylase inhibitor

Somatic embryogenesis is used for vegetative propagation of conifers. Embryogenic cultures can be established from zygotic embryos; however, the embryogenic potential decreases during germination. In Arabidopsis, LEAFY COTYLEDON (LEC) genes are expressed during the embryonic stage, and must be repressed to allow germination. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) causes de-repression of LEC genes. ABSCISIC ACID3 (ABI3) and its Zea mays ortholog VIVIPAROUS1 (VP1) act together with the LEC genes to promote embryo maturation. In this study, we have asked the question whether TSA treatment in a conifer affects the embryogenic potential and the expression of embryogenesis-related genes. We isolated two conifer LEC1-type HAP3 genes, HAP3A and HAP3B, from Picea abies and Pinus sylvestris. A comparative phylogenetic analysis of plant HAP3 genes suggests that HAP3A and HAP3B are paralogous genes originating from a duplication event in the conifer lineage. The expression of HAP3A is high, in both somatic and zygotic embryos, during early embryo development, but decreases during late embryogeny. In contrast, the expression of VP1 is initially low but increases during late embryogeny. After exposure to TSA, germinating somatic embryos of P. abies maintain the competence to differentiate embryogenic tissue, and simultaneously the germination progression is partially inhibited. Furthermore, when embryogenic cultures of P. abies are exposed to TSA during embryo maturation, the maturation process is arrested and the expression levels of PaHAP3A and PaVP1 are maintained, suggesting a possible link between chromatin structure and expression of embryogenesis-related genes in conifers.

Embryogenic potential and expression of embryogenesis-related genes in conifers are affected by treatment with a histone deacetylase inhibitor.

Somatic embryogenesis is used for vegetative propagation of conifers. Embryogenic cultures can be established from zygotic embryos; however, the embryogenic potential decreases during germination. In Arabidopsis, LEAFY COTYLEDON (LEC) genes are expressed during the embryonic stage, and must be repressed to allow germination. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) causes de-repression of LEC genes. ABSCISIC ACID3 (ABI3) and its Zea mays ortholog VIVIPAROUS1 (VP1) act together with the LEC genes to promote embryo maturation. In this study, we have asked the question whether TSA treatment in a conifer affects the embryogenic potential and the expression of embryogenesis-related genes. We isolated two conifer LEC1-type HAP3 genes, HAP3A and HAP3B, from Picea abies and Pinus sylvestris. A comparative phylogenetic analysis of plant HAP3 genes suggests that HAP3A and HAP3B are paralogous genes originating from a duplication event in the conifer lineage. The expression of HAP3A is high, in both somatic and zygotic embryos, during early embryo development, but decreases during late embryogeny. In contrast, the expression of VP1 is initially low but increases during late embryogeny. After exposure to TSA, germinating somatic embryos of P. abies maintain the competence to differentiate embryogenic tissue, and simultaneously the germination progression is partially inhibited. Furthermore, when embryogenic cultures of P. abies are exposed to TSA during embryo maturation, the maturation process is arrested and the expression levels of PaHAP3A and PaVP1 are maintained, suggesting a possible link between chromatin structure and expression of embryogenesis-related genes in conifers.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-011-1418-8) contains supplementary material, which is available to authorized users.

Over-expression of HAP3b enhances primary root elongation in Arabidopsis

HAPs, similar to Heme Activator Proteins (HAP) or nuclear factor-Y (NF-Y) in yeast and animals, play versatile roles in plant growth, development, and responses to environmental cues. HAP3b in Arabidopsis is a member in the HAP3 gene family and is involved in regulating flowering time through the long-day photoperiod pathway (Cai et al., 2007, Plant Physiol 145: 98-105). In this study, we report that overexpression of HAP3b enhances primary root elongation. Detailed analysis showed that HAP3b-overexpression did not affect the length of the root elongation zone and the cell length profiles in the elongation zone. Kinematic analysis indicated that root cells in HAP3b-overexpressors elongate faster than the cells in wild-type roots. Using GUS as a reporter gene, we showed that HAP3b is specifically expressed in the tip region of the root, where cell division and elongation occur. Our results provide evidence to support a role of HAP3b in regulation of root growth.

HapX Positively and Negatively Regulates the Transcriptional Response to Iron Deprivation in Cryptococcus neoformans

The fungal pathogen Cryptococcus neoformans is a major cause of illness in immunocompromised individuals such as AIDS patients. The ability of the fungus to acquire nutrients during proliferation in host tissue and the ability to elaborate a polysaccharide capsule are critical determinants of disease outcome. We previously showed that the GATA factor, Cir1, is a major regulator both of the iron uptake functions needed for growth in host tissue and the key virulence factors such as capsule, melanin and growth at 37°C. We are interested in further defining the mechanisms of iron acquisition from inorganic and host-derived iron sources with the goal of understanding the nutritional adaptation of C. neoformans to the host environment. In this study, we investigated the roles of the HAP3 and HAPX genes in iron utilization and virulence. As in other fungi, the C. neoformans Hap proteins negatively influence the expression of genes encoding respiratory and TCA cycle functions under low-iron conditions. However, we also found that HapX plays both positive and negative roles in the regulation of gene expression, including a positive regulatory role in siderophore transporter expression. In addition, HapX also positively regulated the expression of the CIR1 transcript. This situation is in contrast to the negative regulation by HapX of genes encoding GATA iron regulatory factors in Aspergillus nidulans and Schizosaccharomyces pombe. Although both hapX and hap3 mutants were defective in heme utilization in culture, only HapX made a contribution to virulence, and loss of HapX in a strain lacking the high-affinity iron uptake system did not cause further attenuation of disease. Therefore, HapX appears to have a minimal role during infection of mammalian hosts and instead may be an important regulator of environmental iron uptake functions. Overall, these results indicated that C. neoformans employs multiple strategies for iron acquisition during infection.

Duplication and Functional Diversification of HAP3 Genes Leading to the Origin of the Seed-Developmental Regulatory Gene, LEAFY COTYLEDON1 (LEC1), in Nonseed Plant Genomes

The HAP3 gene encodes a subunit of the CCAAT-box-binding factor (CBF), a highly conserved trimeric activator that recognizes and binds the ubiquitous CCAAT promoter element with high affinity. Two types of HAP3 gene have been identified in plant genomes. The LEAFY COTYLEDON1 (LEC1)-type HAP3 genes encode a functionally specialized subunit of CBF, which is expressed specifically in developing seeds. In contrast, most non-LEC1-type HAP3 genes are expressed in various tissues. It has been proposed that the LEC1-type HAP3 genes originated from the duplication and functional divergence of non-LEC1-type HAP3 genes. However, it is not yet known when this duplication event took place or whether the LEC1-type HAP3 genes appeared at the same time as the origin of seed plants. Here we describe a comprehensive comparison of the duplication patterns of HAP3 genes in different plant genomes. We recognize a major expansion of the HAP3 gene family accompanying the origin and early diversification of land plants and postulate that retrotransposition and other mechanisms of gene duplication have been involved in the expansion of the plant HAP3 gene family. We provide evidence that the LEC1-type HAP3 genes originated in nonseed vascular plant genomes and demonstrate that they are inductively expressed under drought stress in nonseed plants. These genes, however, were recruited to a novel regulatory network in the early stages of seed plant evolution and steadily expressed during seed development and maturation.

Identification, characterization and interaction of HAP family genes in rice

A HAP complex, which consists of three subunits, namely HAP2 (also called NF-YA or CBF-B), HAP3 (NF-YB/CBF-A) and HAP5 (NF-YC/CBF-C), binds to CCAAT sequences in a promoter to control the expression of target genes. We identified 10 HAP2 genes, 11 HAP3 genes and 7 HAP5 genes in the rice genome. All the three HAP family genes encode a protein with a conserved domain in each family and various non-conserved regions in both length and amino acid sequence. These genes showed various expression patterns depending on genes, and various combinations of overlapped expression of the HAP2, HAP3 and HAP5 genes were observed. Furthermore, protein interaction analyses showed interaction of OsHAP3A, a ubiquitously expressed HAP3 subunit of rice, with specific members of HAP5. These results indicate that the formation of specific complex with various HAP subunits combinations can be achieved by both tissue specific expression of three subunit genes and specific interaction of three subunit proteins. This may suggest that the HAP complexes may control various aspects of rice growth and development through tissue specific expression and complex formation of three subunit members.

Identification of new gene expression regulators specifically expressed during plant seed maturation

A cDNA-AFLP approach on Linum usitatissimum (flax) was used to identify genes specifically expressed during the seed maturation process. Among the 20,000 cDNA-AFLP tags produced, 486 were selected for their seed-specific expression during maturation. When compared with the publicly available databases, half of them presented some significant similarity with known plant sequences. The results obtained confirmed the accuracy of the approach as numerous genes previously described as being expressed exclusively in plant seeds were identified in this screen. The focus was on sequences similar to plant regulators involved in the control of gene expression, either at the transcriptional, post-transcriptional, or post-translational levels. Using a real-time RT-PCR approach, seed-specific expression kinetics were confirmed for 13 of these regulators that were never characterized for being expressed during seed maturation. Among these, a flax gene of the non-LEC1-like HAP3 family and a flax MYB factor were shown to be expressed in specialized tissues of flax embryo using an in situ hybridization approach. By expression kinetic comparison between these flax genes and their Arabidopsis counterparts, it was found that the new HAP3 gene should be related to a ubiquitous seed maturation mechanism, while a new MYB factor appears to be related to a more seed-specific maturation mechanism. These results demonstrate the utility of the flax database in not only identifying new genes expressed during seed maturation but also in being able to highlight the distinction between conserved and non-conserved seed maturation mechanisms.

The Hypocrea jecorina HAP 2/3/5 protein complex binds to the inverted CCAAT-box (ATTGG) within the cbh2 (cellobiohydrolase II-gene) activating element.

The 5' regulatory region of the chh2 gene, encoding cellobiohydrolase II, of the filamentous fungus Hypocrea jecorina contains the cbh2 activating element (CAE) which is essential for cbh2 expression. The CAE consists of two separate, adjacent motifs, a CCAAT box on the template strand (ATTGG) and a GTAATA box on the coding strand, which co-operate in the induction of the gene by cellulose or sophorose. EMSA supershift experiments using an antibody against Aspergillus nidulans HAPC suggested that the complex which binds to the H. jecorina CCAAT box contains a HAPC homolog. To obtain direct evidence for this, we have cloned the hap2, hap3 and hap5 genes from H. jecorina. They encode proteins whose core regions display great similarity to Aspergillus HAPB, HAPC and HAPE and to known HAP homologs from other organisms. All three genes are transcribed in a carbon source-independent manner. A. nidulans deltahap strains were functionally complemented in vitro by the overexpressed H. jecorina HAP2, HAP3 and HAP5 proteins, and they thus represent subunits of the CCAAT-binding complex. Furthermore, all three proteins (HAP2, HAP3 and HAP5) were needed to bind to the CAE in the H. jecorina cbh2 gene promoter in vitro. We conclude that the CCAAT box on the template strand in CAE is bound by the H. jecorina equivalent of the HAP protein complex.

Genetic aspects of carbon catabolite repression of the STA2 glucoamylase gene in Saccharomyces cerevisiae.

Three regulatory genes, known to be required for glucose repression/derepression of some genes in Saccharomyces cerevisiae, were disrupted to study their effects on the carbon-source regulation of the STA2 glucoamylase gene expression. Using a STA2-lacZ fusion it was found that: (1) the MIG1 gene is dispensable for the repression of the STA2 gene; (2) there are two components in the carbon-source repression of STA2: HXK2-dependent and HXK2-independent; and (3) the HAP2 gene seems to be involved in repression rather than activation of the STA2 expression.

Transcriptional regulation of the KlDLD gene, encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase in Kluyveromyces lactis : effect of Klhap2 and fog mutations

Expression of the Kluyveromyces lactis KlDLD gene, encoding the mitochondrial enzyme D-lactate ferricytochrome c oxidoreductase (D-LCR), is subject to two metabolic controls at the transcriptional level: induction by lactate, the substrate of the D-LCR enzyme, and repression by glucose. By Northern analysis we determined the kinetics of the two regulatory processes and, by measurement of the expression of LacZ gene fused to the KlDLD promoter, we identified cis-elements involved in glucose repression and lactate induction. The effect of trans-acting factors on the transcription of KlDLD has been analyzed. The KlDLD gene is controlled by the products of the FOG1 and FOG2 genes, previously identified as involved in glucose de-repression. Moreover, the KlDLD gene is regulated by the product of KlHAP2, homologous to the HAP2 gene which in Saccharomyces cerevisiae is required for the induction of genes encoding mitochondrial components, upon shifting from a fermentable to a non-fermentable carbon source. We have demonstated that the KlHAP2 gene is necessary both for the lactate induction of KlDLD mRNA synthesis and for growth on this oxidative carbon source.

Genetic aspects of carbon catabolite repression of the STA2 glucoamylase gene in Saccharomyces cerevisiae

Three regulatory genes, known to be required for glucose repression/derepression of some genes in Saccharomyces cerevisiae, were disrupted to study their effects on the carbon-source regulation of the STA2 glucoamylase gene expression. Using a STA2-lacZ fusion it was found that: (1) the MIG1 gene is dispensable for the repression of the STA2 gene; (2) there are two components in the carbon-source repression of STA2: HXK2-dependent and HXK2-independent; and (3) the HAP2 gene seems to be involved in repression rather than activation of the STA2 expression.

The hapC gene of Aspergillus nidulans is involved in the expression of CCAAT-containing promoters.

The 5' regulatory region of the amdS gene of Aspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence which is required for setting the basal level of amdS expression. Mobility shift studies have identified a factor in A. nidulans nuclear extracts which binds to this CCAAT sequence. In Saccharomyces cerevisiae the HAP3 gene encodes one component of a multisubunit complex that binds CCAAT sequences. A search of the EMBL and SwissProt databases has revealed an A. nidulans sequence with significant homology to the HAP3 gene adjacent to the previously cloned regulatory gene amdR. Sequencing of the remainder of this region has confirmed the presence of a gene, designated hapC, with extensive homology to HAP3. The predicted amino acid sequence of HapC shows extensive identity to HAP3 in the central conserved domain, but shows little conservation in the flanking sequences. A haploid carrying a hapC deletion has been created and is viable, but grows poorly on all media tested. This null mutant grows especially slowly on acetamide as a sole carbon and nitrogen source, indicating that hapC plays a role in amdS expression. In agreement with this notion, it has been shown that the hapC deletion results in reduced levels of expression of an amdS::lacZ reporter gene and this effect is particularly evident under conditions of carbon limitation. Nuclear extracts prepared from the hapC deletion mutant show no CCAAT binding activity to the amdS or gatA promoters, indicating that hapC may encode a component of the complex binding at this sequence.

Dual influence of the yeast Cat1p (Snf1p) protein kinase on carbon source-dependent transcriptional activation of gluconeogenic genes by the regulatory gene CAT8.

The CSRE (carbon source-responsive element) is a sequence motif responsible for the transcriptional activation of gluconeogenic structural genes in Saccharomyces cerevisiae. We have isolated a regulatory gene, DIL1 (derepression of isocitrate lyase, = CAT8), which is specifically required for derepression of CSRE-dependent genes. Expression of CAT8 is carbon source regulated and requires a functional Cat1p (Snf1p) protein kinase. The derepression defect of CAT8 in a cat1 mutant could be suppressed by a mutant Mig1p repressor protein. Derepression of CAT8 also requires a functional HAP2 gene, suggesting a regulatory connection between respiratory and gluconeogenic genes. Carbon source-dependent protein-CSRE complexes detected in a gel retardation analysis with wild-type extracts were absent in cat8 mutant extracts. However, similar experiments with an epitope-tagged CAT8 gene product in the presence of tag-specific antibodies gave evidence against a direct binding of Cat8p to the CSRE. A constitutively expressed GAL4-CAT8 fusion gene revealed a carbon source-dependent transcriptional activation of a UAS(GAL)-containing reporter gene. Activation mediated by Cat8p was no longer detectable in a cat1 mutant. Thus, biosynthetic control of CAT8 as well as transcriptional activation by Cat8p requires a functional Cat1p protein kinase. A model proposing CAT8 as a specific activator of a transcription factor(s) binding to the CSRE is discussed.