Differential Expression of the Arabidopsis Nia1 and Nia2 Genes1 : Cytokinin-Induced Nitrate Reductase Activity Is Correlated With Increased Nia1 Transcription and mRNA Levels

Nitrate reductase (NR) activity increased up to 14-fold in response to treatment of Arabidopsis thaliana seedlings with the cytokinin benzyladenine. NR induction was observed in seedlings germinated directly on cytokinin-containing medium, seedlings transferred to cytokinin medium, and seedlings grown in soil in which cytokinin was applied directly to the leaves. About the same level of induction was seen in both wild-type and Nia2-deletion mutants, indicating that increased NR activity is related to the expression of the minor NR gene, Nia1. The steady-state Nia1 mRNA level was increased severalfold in both wild-type and mutant seedlings after benzyladenine treatment. Transcript levels of the Nia2 gene, which is responsible for 90% of the NR activity in developing wild-type seedlings, did not show any changes upon cytokinin treatment. Nuclear run-on assays demonstrated that Nia1 gene transcription increased dramatically after cytokinin treatment.

Two Isoforms of NADPH:Cytochrome P450 Reductase in Arabidopsis thaliana : Gene Structure, Heterologous Expression in Insect Cells, and Differential Regulation

We have investigated two NADPH-cytochrome (Cyt) P450 reductase isoforms encoded by separate genes (AR1 and AR2) in Arabidopsis thaliana. We isolated AR1 and AR2 cDNAs using a mung bean (Phaseolus aureus L.) NADPH-Cyt P450 reductase cDNA as a probe. The recombinant AR1 and AR2 proteins produced using a baculovirus expression system showed similar Km values for Cyt c and NADPH, respectively. In the reconstitution system with a recombinant cinnamate 4-hydroxylase (CYP73A5), the recombinant AR1 and AR2 proteins gave the same level of cinnamate 4-hydroxylase activity (about 70 nmol min−1 nmol−1 P450). The AR2 gene expression was transiently induced by 4- and 3-fold within 1 h of wounding and light treatments, respectively, and the induction time course preceded those of CYP73A5 and a phenylalanine ammonia-lyase (PAL1) gene. On the contrary, the AR1 expression level did not change during the treatments. Analysis of the AR1 and AR2 gene structure revealed that only the AR2 promoter contained three putative sequence motifs (boxes P, A, and L), which are involved in the coordinated expression of CYP73A5 and other phenylpropanoid pathway genes. These results suggest the possibility that AR2 transcription may be functionally linked to the induced levels of phenylpropanoid pathway enzymes.

Differential expression of the ToxR regulon in classical and E1 Tor biotypes of Vibrio cholerae is due to biotype-specific control over toxT expression.

The two major disease-causing biotypes of Vibrio cholerae, classical and El Tor, exhibit differences in their epidemic nature. Their behavior in the laboratory also differs in that El Tor strains produce two major virulence factors, cholera toxin (CT) and the toxin coregulated pilus (TCP), only under very restricted growth conditions, whereas classical strains do so in standard laboratory medium. Expression of toxin and TCP is controlled by two activator proteins, ToxR and ToxT, that operate in cascade fashion with ToxR controlling the synthesis of ToxT. Both biotypes express equivalent levels of ToxR, but only classical strains appear to express ToxT when grown in standard medium. In this report we show that restrictive expression of CT and TCP can be overcome in El Tor strains by expressing ToxT independently of ToxR. An El Tor strain lacking functional ToxT does not express CT or TCP, ruling out existence of a cryptic pathway for virulence regulation in this biotype. These results may have implications for understanding the evolution of El Tor strains toward reduced virulence with respect to classical strains.

Chromosomal localization of the mammalian peptide-methionine sulfoxide reductase gene and its differential expression in various tissues.

Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous protein that can reduce methionine sulfoxide residues in proteins as well as in a large number of methyl sulfoxide compounds. The expression of MsrA in various rat tissues was determined by using immunocytochemical staining. Although the protein was found in all tissues examined, it was specifically localized to renal medulla and retinal pigmented epithelial cells, and it was prominent in neurons and throughout the nervous system. In addition, blood and alveolar macrophages showed high expression of the enzyme. The msrA gene was mapped to the central region of mouse chromosome 14, in a region of homology with human chromosomes 13 and 8p21.

A mechanism for the differential regulation of gonadotropin subunit gene expression by gonadotropin-releasing hormone.

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) is released in a pulsatile fashion, with its frequency varying throughout the reproductive cycle. Varying pulse frequencies and amplitudes differentially regulate the biosynthesis and secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by pituitary gonadotropes. The mechanism by which this occurs remains a major question in reproductive physiology. Previous studies have been limited by lack of available cell lines that express the LH and FSH subunit genes and respond to GnRH. We have overcome this limitation by transfecting the rat pituitary GH3 cell line with rat GnRH receptor (GnRHR) cDNA driven by a heterologous promoter. These cells, when cotransfected with regulatory regions of the common alpha, LH beta, or FSH beta subunit gene fused to a luciferase reporter gene, respond to GnRH with an increase in luciferase activity. Using this model, we demonstrate that different cell surface densities of the GnRHR result in the differential regulation of LH and FSH subunit gene expression by GnRH. This suggests that the differential regulation of gonadotropin subunit gene expression by GnRH observed in vivo in rats may, in turn, be mediated by varying gonadotrope cell surface GnRHR concentrations. This provides a physiologic mechanism by which a single ligand can act through a single receptor to regulate differentially the production of two hormones in the same cell.

Identification of the human prostatic carcinoma oncogene PTI-1 by rapid expression cloning and differential RNA display.

Elucidating the relevant genomic changes mediating development and evolution of prostate cancer is paramount for effective diagnosis and therapy. A putative dominant-acting nude mouse prostatic carcinoma tumor-inducing gene, PTI-1, has been cloned that is expressed in patient-derived human prostatic carcinomas but not in benign prostatic hypertrophy or normal prostate tissue. PTI-1 was detected by cotransfecting human prostate carcinoma DNA into CREF-Trans 6 cells, inducing tumors in nude mice, and isolating genes displaying increased expression in tumor-derived cells by using differential RNA display (DD). Screening a human prostatic carcinoma (LNCaP) cDNA library with a 214-bp DNA fragment found by DD permitted the cloning of a full-length 2.0-kb PTI-1 cDNA. Sequence analysis indicates that PTI-1 is a gene containing a 630-bp 5' sequence and a 3' sequence homologous to a truncated and mutated form of human elongation factor 1 alpha. In vitro translation demonstrates that the PTI-1 cDNA encodes a predominant approximately 46-kDa protein. Probing Northern blots with a DNA fragment corresponding to the 5' region of PTI-1 identifies multiple PTI-1 transcripts in RNAs from human carcinoma cell lines derived from the prostate, lung, breast, and colon. In contrast, PTI-1 RNA is not detected in human melanoma, neuroblastoma, osteosarcoma, normal cerebellum, or glioblastoma multiforme cell lines. By using a pair of primers recognizing a 280-bp region within the 630-bp 5' PTI-1 sequence, reverse transcription-PCR detects PTI-1 expression in patient-derived prostate carcinomas but not in normal prostate or benign hypertrophic prostate tissue. In contrast, reverse transcription-PCR detects prostate-specific antigen expression in all of the prostate tissues. These results indicate that PTI-1 may be a member of a class of oncogenes that could affect protein translation and contribute to carcinoma development in human prostate and other tissues. The approaches used, rapid expression cloning with the CREF-Trans 6 system and the DD strategy, should prove widely applicable for identifying and cloning additional human oncogenes.

Restriction fragment length polymorphism-coupled domain-directed differential display: a highly efficient technique for expression analysis of multigene families.

In this paper, a reverse-transcriptase PCR-based protocol suitable for efficient expression analysis of multigene families is presented. The method combines restriction fragment length polymorphism (RFLP) technology with a gene family-specific version of mRNA differential display and hence is called "RFLP-coupled domain-directed differential display. "With this method, expression of all members of a multigene family at many different developmental stages, in diverse tissues and even in different organisms, can be displayed on one gel. Moreover, bands of interest, representing gene family members, are directly accessible to sequence analysis, without the need for subcloning. The method thus enables a detailed, high-resolution expression analysis of known gene family members as well as the identification and characterization of new ones. Here the technique was used to analyze differential expression of MADS-box genes in male and female inflorescences of maize (Zea mays ssp. mays). Six different MADS-box genes could be identified, being either specifically expressed in the female sex or preferentially expressed in male or female inflorescences, respectively. Other possible applications of the method are discussed.

Differential modulation of Th1- and Th2-related cytokine mRNA expression by a synthetic peptide homologous to a conserved domain within retroviral envelope protein.

The influence of a synthetic retroviral peptide, CKS-17, on T helper type 1 (Th1)- or Th2-related cytokines was investigated in human blood mononuclear cells. Cells were stimulated with staphylococcal enterotoxin A, anti-CD3 plus anti-CD28 monoclonal antibodies, or lipopolysaccharide to induce cytokine mRNA. mRNA was detected by a reverse transcription-polymerase chain reaction or Northern blot analysis. CKS-17 down-regulated stimulant-induced mRNA accumulation for interferon gamma (IFN-gamma), interleukin (IL)-2, and p40 heavy and p35 light chains of IL-12, a cytokine that mediates development of Th1 response. CKS-17 up-regulated stimulant-induced mRNA accumulation of IL-10 and did not suppress Th2-related cytokine (IL-4, IL-5, IL-6, or IL-13) mRNA expression. A reverse sequence of CKS-17 peptide, used as a control, showed no such action. Anti-human IL-10 monoclonal antibody blocked ability of CKS-17 to inhibit mRNA accumulation for IFN-gamma but not the CKS-17 suppressive activity of IL-12 p40 heavy chain mRNA. Thus, CKS-17-mediated suppression of IFN-gamma mRNA expression is dependent upon augmentation of IL-10 production by CKS-17. This conserved component of several retroviral envelope proteins, CKS-17, may act as an immunomodulatory epitope responsible for cytokine dysregulation that leads to suppression of cellular immunity.

The FEZ1 gene at chromosome 8p22 encodes a leucine-zipper protein, and its expression is altered in multiple human tumors

Alterations of human chromosome 8p occur frequently in many tumors. We identified a 1.5-Mb common region of allelic loss on 8p22 by allelotype analysis. cDNA selection allowed isolation of several genes, including FEZ1. The predicted Fez1 protein contained a leucine-zipper region with similarity to the DNA-binding domain of the cAMP-responsive activating-transcription factor 5. RNA blot analysis revealed that FEZ1 gene expression was undetectable in more than 60% of epithelial tumors. Mutations were found in primary esophageal cancers and in a prostate cancer cell line. Transcript analysis from several FEZ1-expressing tumors revealed truncated mRNAs, including a frameshift. Alteration and inactivation of the FEZ1 gene may play a role in various human tumors.

HLA-G expression in melanoma: A way for tumor cells to escape from immunosurveillance

Considering the well established role of nonclassical HLA-G class I molecules in inhibiting natural killer (NK) cell function, the consequence of abnormal HLA-G expression in malignant cells should be the escape of tumors from immunosurveillance. To examine this hypothesis, we analyzed HLA-G expression and NK sensitivity in human malignant melanoma cells. Our analysis of three melanoma cell lines and ex vivo biopsy demonstrated that (i) IGR and M74 human melanoma cell lines exhibit a high level of HLA-G transcription with differential HLA-G isoform transcription and protein expression patterns, (ii) a higher level of HLA-G transcription ex vivo is detected in a skin melanoma metastasis biopsy compared with a healthy skin fragment from the same individual, and (iii) HLA-G protein isoforms other than membrane-bound HLA-G1 protect IGR from NK lysis. It thus appears of critical importance to consider the specific role of HLA-G expression in tumors in the design of future cancer immunotherapies.

Differential roles of Ca2+/calmodulin-dependent kinases in posttetanic potentiation at input selective glutamatergic pathways

The electrosensory lateral line lobe (ELL) of the electric fish Apteronotus leptorhynchus is a layered medullary region receiving electroreceptor input that terminates on basal dendrites of interneurons and projection (pyramidal) cells. The molecular layer of the ELL contains two distinct glutamatergic feedback pathways that terminate on the proximal (ventral molecular layer, VML) and distal (dorsal molecular layer) apical dendrites of pyramidal cells. Western blot analysis with an antibody directed against mammalian Ca2+/calmodulin-dependent kinase 2, α subunit (CaMK2α) recognized a protein of identical size in the brain of A. leptorhynchus. Immunohistochemistry demonstrated that CaMK2 α expression in the ELL was restricted to fibers and terminals in the VML. Posttetanic potentiation (PTP) could be readily elicited in pyramidal cells by stimulation of either VML or DML in brain slices of the ELL. PTP in the VML was blocked by extracellular application of a CaMK2 antagonist (KN62) while intracellular application of KN62 or a CaMK2 inhibitory peptide had no effect, consistent with the presynaptic localization of CaMK2 α in VML. PTP in the dorsal molecular layer was not affected by extracellular application of KN62. Anti-Hebbian plasticity has also been demonstrated in the VML, but was not affected by KN62. These results demonstrate that, while PTP can occur independent of CaMK2, it is, in some synapses, dependent on this kinase.

Mouse mammary tumor virus/v-Ha-ras transgene-induced mammary tumors exhibit strain-specific allelic loss on mouse chromosome 4

Hybrid mice carrying oncogenic transgenes afford powerful systems for investigating loss of heterozygosity (LOH) in tumors. Here, we apply this approach to a neoplasm of key importance in human medicine: mammary carcinoma. We performed a whole genome search for LOH using the mouse mammary tumor virus/v-Ha-ras mammary carcinoma model in female (FVB/N × Mus musculus castaneus)F1 mice. Mammary tumors developed as expected, as well as a few tumors of a second type (uterine leiomyosarcoma) not previously associated with this transgene. Genotyping of 94 anatomically independent tumors revealed high-frequency LOH (≈38%) for markers on chromosome 4. A marked allelic bias was observed, with M. musculus castaneus alleles almost exclusively being lost. No evidence of genomic imprinting effects was noted. These data point to the presence of a tumor suppressor gene(s) on mouse chromosome 4 involved in mammary carcinogenesis induced by mutant H-ras expression, and for which a significant functional difference may exist between the M. musculus castaneus and FVB/N alleles. Provisional subchromosomal localization of this gene, designated Loh-3, can be made to a distal segment having syntenic correspondence to human chromosome 1p; LOH in this latter region is observed in several human malignancies, including breast cancers. Evidence was also obtained for a possible second locus associated with LOH with less marked allele bias on proximal chromosome 4.

Expression of an ortholog of replication protein A1 (RPA1) is induced by gibberellin in deepwater rice

Internodes of deepwater rice are induced to grow rapidly when plants become submerged. This adaptation enables deepwater rice to keep part of its foliage above the rising flood waters during the monsoon season and to avoid drowning. This growth response is, ultimately, elicited by the plant hormone gibberellin (GA). The primary target tissue for GA action is the intercalary meristem of the internode. Using differential display of mRNA, we have isolated a number of genes whose expression in the intercalary meristem is regulated by GA. The product of one of these genes was identified as an ortholog of replication protein A1 (RPA1). RPA is a heterotrimeric protein involved in DNA replication, recombination, and repair and also in regulation of transcription. A chimeric construct, in which the single-stranded DNA-binding domain of rice RPA1 was spliced into the corresponding region of yeast RPA1, was able to complement a yeast rpa1 mutant. The transcript level of rice RPA1 is high in tissues containing dividing cells. RPA1 mRNA levels increase rapidly in the intercalary meristem during submergence and treatment with GA before the increase in the level of histone H3 mRNA, a marker for DNA replication.

ETS target genes: Identification of Egr1 as a target by RNA differential display and whole genome PCR techniques

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing’s sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II, and Egr1 genes. The ETS binding sites derived from these three regulatory sequences showed specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene.

Soldier caste-specific gene expression in the mandibular glands of Hodotermopsis japonica (Isoptera: Termopsidae)

Although “polymorphic castes” in social insects are well known as one of the most important phenomena of polyphenism, few studies of caste-specific gene expressions have been performed in social insects. To identify genes specifically expressed in the soldier caste of the Japanese damp-wood termite Hodotermopsis japonica, we employed the differential-display method using oligo(dT) and arbitrary primers, compared mRNA from the heads of mature soldiers and pseudergates (worker caste), and identified a clone (PCR product) 329 bp in length termed SOL1. Northern blot analysis showed that the SOL1 mRNA is about 1.0 kb in length and is expressed specifically in mature soldiers, but not in pseudergates, even in the presoldier induction by juvenile hormone analogue, suggesting that the product is specific for terminally differentiated soldiers. By using the method of 5′- and 3′-rapid amplification of cDNA ends, we isolated the full length of SOL1 cDNA, which contained an ORF with a putative signal peptide at the N terminus. The sequence showed no significant homology with any other known protein sequences. In situ hybridization analysis showed that SOL1 is expressed specifically in the mandibular glands. These results strongly suggest that the SOL1 gene encodes a secretory protein specifically synthesized in the mandibular glands of the soldiers. Histological observations revealed that the gland actually develops during the differentiation into the soldier caste.