The c-terminal DNA-binding domain ofChironomus BR gene products shows preferential affinity for (dA,dT)-rich sequences

Balbiani ring genes (BRs), the most active loci in the polytene chromosomes of the salivary gland of the midgeChironomus (Diptera), code for secretory giant peptides (the sp-I family). Evidence previously reported indicated that the conserved C-terminal region of proteins of the sp-I family had DNA-binding properties (assayed with sp-Ia), and one such region, derived fromBR2.2, which codes for the product sp-Ib, might occur as a stable independent peptide, being transferred to the nucleus where it is detectable in the largeBRs (BR1 andBR2), among other structures, by immunostaining. Here, we show that the C-terminal portion of one of theBR gene products, expressed as a glutathione-S-transferase fusion protein shows preferential affinity for A.T-rich sequences and binds with varying affinity to restriction fragments of the A.T-rich BR1 promoter. The binding was inhibited by distamycin, suggesting that the interaction involves the minor groove of the DNA. Analysis of the promoter fragments by gel electrophoresis indicated that most appeared to present a conspicuous bend, as deduced from their anomalous electrophoretic mobilities. Furthermore, the affinity of the C-terminal domain for the different promoter fragments appeared to correlate with the degree of bending. Thus, the C-terminal domain might play a role in controlling gene expression by binding to A.T-rich sequences, including those of theBR genes.     

Nuclear localization of a DNA-binding C-terminal domain from Balbiani ring coded secretory protein.

All known Balbiani ring (BR) genes in Chironomus tentans, coding for giant secretory proteins, the sp-I family, end with a short (110 codons) 3'-end exon which is highly conserved in evolution and is structurally unrelated to the sequences characterizing the core of these proteins. We find that the expressed product, the C-terminal domain, shows sequence-specific DNA binding and that it is likely to be absent in one of the sp-I components, sp-Ib, believed to be coded by the BR2.2 gene. Immunohistochemistry shows that material with reactivity towards antibody against the C-terminal domain is present in the nuclei, and specifically enriched in Balbiani ring 1 and 2. Western blotting of extracts from isolated nuclei demonstrates a component with the same antibody reactivity and of an apparent size somewhat larger than that of the domain. The possibility is discussed that the C-terminal part, which is part of the secretion when derived from some of the BR genes, might be cleaved off and function as a feedback signal to control BR gene activity when derived from the BR2.2 gene.     

Individual variations in the content of giant secretory polypeptides in salivary glands of Chironomus

Salivary glands in aquatic larvae of Chironomus are responsible for formation of a fiber that larvae use to construct feeding tubes. Major constituents of this fiber include a family (the sp-I family) of high M _r (1 × 10⁶) secretory polypeptides. Because of our interest in the polypeptide composition and polymerization of the salivary fiber we conducted a survey of the electrophoretic pattern of sp-I components found in salivary glands obtained from individual larvae. The survey encompassed ten strains of Chironomus tentans, three strains of Chironomus pallidivittatus and four strains of Chironomus thummi. Salivary glands from C. tentans and C. pallidivittatus contained at least four sp-I components (sp-Ia, sp-Ib, sp-Ic and sp-Id) that behave identically with regard to their electrophoretic mobility and detectability when larvae were exposed to galactose or glycerol. Sp-I components in C. thummi were generally fewer and not directly comparable by electrophoretic mobility to sp-I components in the other two species. During this survey two important alterations were observed in the electrophoretic pattern of sp-I components obtained from C. tentans and C. pallidivittatus. First, all four sp-I components exhibited, with a low frequency, double bands that appeared as slow-versus-fast electrophoretic variants of a particular component. Secondly, the relative steady-state level of each sp-I component fluctuated in comparison to other sp-I components in the same extract. This fluctuation varied such that any one sp-I component might appear as a single prominent component. Sp-I components are encoded by a multigene family located in Balbiani rings (BRs). Results presented here, in conjunction with known nucleotide sequence data from BR genes, led us to the following conclusions. The slow and fast electrophoretic variants observed for each sp-I component suggest that each corresponding BR gene may be able to expand and/or contract in size. The observed degree of independent fluctuation in the steady-state level of each sp-I component suggests that each BR gene may be able to regulate its expression independently from the others. Finally, the observation that salivary glands sometimes contained only one prominent sp-I component led us to hypothesize that, whereas salivary fibers might typically be heteropolymers comprised of two or more types of sp-I components, homopolymers comprised of only one sp-I component may also exist.     

Identification of novel members reveals the structural and functional divergence of lepidopteran-specific Lipoprotein_11 family

30K proteins (30KPs) are classified into the lepidopteran-specific Lipoprotein_11 family. They are involved in various physiological processes such as energy storage, embryonic development, and immune response in the silkworm. To date, 30KPs were only found in Bombyx mori and Manduca sexta. Moreover, the C-termini of ENF peptide binding proteins (ENF-BPs) show similarity to 30KPs. ENF peptides are multifunctional insect cytokines and involved in growth regulation and defense reaction, whereas ENF-BPs act as active regulators of ENF peptides. In order to get insights into this gene family in Lepidoptera, we performed an extensive survey of lepidopteran-derived genome and EST datasets. We identified 73 30KP homologous genes in 12 lepidopteran species, of which 56 are novel members. The structural and phylogenetic analyses revealed that these genes could be classified into three groups: ENF-BP genes, typical 30KP genes, and serine/threonine-rich 30KP (S/T-rich 30KP) genes. The C-terminal regions are common to all the three subfamilies, but the N-termini are highly variable. We found a novel subfamily of Lipoprotein_11 and named it S/T-rich 30KP according to its exclusive S/T-rich domain in the N terminus. ENF-BP was also found to contain a special domain in the N terminus, which is homologous to Pp-0912 of Pseudomonas putida. Microarray data and semi-quantitative RT-PCR showed that the three groups have their respective temporal–spatial expression patterns. S/T-rich 30KP genes have enriched expression in the mature testis and might be involved in spermiogenesis or fertilization. Typical 30KP genes are expressed mainly in the fat body and integument at the larvae and pupae stages. ENF-BP genes are expressed predominantly in the hemocyte. The differential spatial–temporal expression profiles revealed the functional divergence of three Lipoprotein_11 subfamilies.     

Domain Structure of the Prokaryotic Selenocysteine-specific Elongation Factor SelB

Incorporation of the non-canonical amino acid selenocysteine into proteins requires the activity of the elongation factor SelB which substitutes for the function of EF-Tu. In contrast to EF-Tu, SelB binds selenocystylated tRNA^Secand an mRNA secondary structure adjacent to the UGA selenocysteine codon. To gain information on the domain structure of this specialized translation factor, theselBgenes from two bacteria unrelated toEscherichia coli(Clostridium thermoaceticumandDesulfomicrobium baculatum) were cloned and sequenced. The derived amino acid residue sequences were compared to those of SelB fromE. coliandHaemophilus influenzaeand to EF-Tu sequences. The alignment revealed that SelB contains all three domains characterized for EF-Tu. A fourth, C-terminally located domain shows only limited sequence conservation within the four SelB proteins. To elucidate the function of this C-terminal part a structure-function analysis of SelB fromE. coliwas performed. It showed that a C-terminal 17 kDa subdomain of the translation factor, when expressed separately, specifically binds the mRNA secondary structure. The recognition motif itself could be reduced to a 17 nucleotide minihelix without loss of binding affinity and specificity. A truncated SelB lacking the mRNA binding domain was still able to interact with selenocysteyl-tRNA^Sec. Expression of the mRNA binding domain alone suppressed selenocysteine insertionin vivoby competing with SelB for its binding site at the mRNA. The results indicate that SelB can be considered as an EF-Tu homolog hooked to the mRNAviaits C-terminal domain.     

Phosphate incorporation into secretory protein of Chironomus salivary glands occurs during translation

The Balbiani rings (BR) in Chironomus salivary gland cells code for giant secretory proteins, the sp-I family. During normal growth conditions the phosphorylated proteins sp-Ia and sp-Ib are formed with most phosphate present as phosphoserine. We can show that most if not all incorporation of ³²P into sp-I occurs in parallel with the incorporation of [³⁵S]-methionine in the giant polysomes that form sp-I and contain BR-derived mRNA. We suggest that the main function of phosphorylation of sp-Ia and sp-Ib is to provide charge neutralization of an excess of lysine and arginine residues and is therefore required during early stages of protein folding. This view is supported by the previous observation that glutamic (and aspartic) acid largely substitute for phosphoserine in a non-phosphorylated member of the sp-I family, sp-Ic, which is produced during phosphate starvation.     

The affinity of different MBD proteins for a specific methylated locus depends on their intrinsic binding properties

The methyl-CpG binding domain (MBD) family of proteins was defined based on sequence similarity in their DNA binding domains. In light of their high degree of conservation, it is of inherent interest to determine the genomic distribution of these proteins, and their associated co-repressor complexes. One potential determinant of specificity resides in differences in the intrinsic DNA binding properties of the various MBD proteins. In this report, we use a capillary electrophoretic mobility shift assay (CEMSA) with laser-induced fluorescence (LIF) and neutral capillaries to calculate MBD–DNA binding affinities. MBD proteins were assayed on pairs of methylated and unmethylated duplex oligos corresponding to the promoter regions of the BRCA1, MLH1, GSTP1 and p16^INK4a genes, and binding affinities for each case were calculated by Scatchard analyses. With the exception of mammalian MBD3 and Xenopus MBD3 LF, all the MBD proteins showed higher affinity for methylated DNA (in the nanomolar range) than for unmethylated DNA (in the micromolar range). Significant differences between MBD proteins in the affinity for methylated DNA were observed, ranging within two orders of magnitude. By mutational analysis of MBD3 and using CEMSA, we demonstrate the critical role of specific residues within the MBD in conferring selectivity for methylated DNA. Interestingly, the binding affinity of specific MBD proteins for methylated DNA fragments from naturally occurring sequences are affected by local methyl-CpG spacing.     

On the Origin and Evolution of Plant Brassinosteroid Receptor Kinases

Brassinosteroid (BR) signaling pathway is so far the best-understood receptor-kinase signaling pathway in plants. In Arabidopsis, the activation of this pathway requires binding of BRs to the receptor kinase BRASSINOSTEROID-INSENSITIVE I (AtBRI1). Although the function of AtBRI1 has been extensively studied, it is not known when the binding function emerged and how this important component of BR signaling pathway and related genes (the BRI1–BRL gene family) have evolved in plants. We define BRI1–BRL genes in sequenced plant genomes, construct profiles for critical protein domains, scan them against all accessible plant gene/EST resources, and reveal the evolution of domain configuration of this family. We also investigate its evolutionary pattern through phylogenetic analysis. The complete BR receptor domain configuration originates through two domain gain events in the ancestral receptor-like kinase: first juxtamembrane domain gained during the early diversification of land plants, and then island domain (ID) acquired in the common ancestor of angiosperms and gymnosperms after its divergence from spike moss. The 70 amino acid ID has characteristic sequences of BRI1–BRL family and this family keeps relative stable copy numbers during the history of angiosperms and the majority of duplications and losses have occurred in terminal taxa in current taxon sampling. This study reveals important events shaping structural and functional characteristics of plant BR receptors. It answers the question of how and when BR receptors originates, which provide insights into the origin and evolution of the BR signaling pathway.     

Correlated changes in steady-state levels of Balbiani ring mRNAs and secretory polypeptides in salivary glands of Chironomus tentans

Balbiani rings (BRs) on polytenized chromosomes in Chironomid salivary glands contain members of a homologous multigene family that encodes a family (the sp-I family) of high M _r secretory polypeptides. Each of these BR genes is comprised largely of tandemly duplicated core repeat sequences consisting of related constant (C) regions and intergenically divergent subrepeat (SR) regions. A set of oligodeoxyribonucleotide probes were synthesized that correspond to the transcribed strand of the SR region of BR1, BR2, BR2/, and BR6 core repeats. Under a defined set of conditions, it was possible to show that each oligonucleotide probe hybridized exclusively to its cognate repeat type without hybridization to other repeat types in cloned DNA templates. These BR probes were then used in dotblot hybridization experiments to simultaneously follow alterations in the steady-state level of BR mRNAs in response to prolonged exposure of larvae to galactose. The results indicated that the relative amounts of these four BR mRNAs may change in a noncoordinate manner. These BR probes were also used in experiments to compare simultaneously the salivary gland content of sp-I components and specific BR mRNAs in larvae that exhibited naturally occurring or induced alterations in BR gene expression. A correlation was found which suggested that sp-Ia is encoded in a gene comprised of BR1 repeats, sp-Ib is encoded by BR2 repeats, sp-Ic is encoded by BR6 repeats and sp-Id is encoded by BR2 repeats.     

Use of a short A/T-rich cassette for enhanced expression of cloned genes inEscherichia coli

A short (43-bp) A/T-rich stretch of DNA located in The intergenic region between thebaiA2 andbaiF genes fromEubacterium sp. strain VPI 12708 was amplified by polymerase chain reaction (PCR) and inserted in front of the Shine-Dalgarno (SD) sequences of three inefficiently-expressedEubacterium sp. strain VPI 12708 genes cloned inEschcrichia coli plasmids. Insertion of this A/T-rich cassette increased gene expression in all cases tested. Deletion of part of the A/T-rich region from abaiF clone in pUC19 resulted in decreased gene expression. Synthesis of specific mRNA was increased with addition of the A/T-rich cassette to constructs containing thebaiC gene from Eubacterium sp. strain VPI 12708, but mRNA synthesis was not significantly changed in cells containing plasmid constructs with thebaiF andbaiG genes. Enhanced translation resulting from a decrease in mRNA secondary structure in the ribosome binding site region is discussed as a possible reason for increased gene expression with the A/T-rich cassette.     

High-Mobility-Group A-Like CarD Binds to a DNA Site Optimized for Affinity and Position and to RNA Polymerase To Regulate a Light-Inducible Promoter in Myxococcus xanthus

The CarD-CarG complex controls various cellular processes in the bacterium Myxococcus xanthus including fruiting body development and light-induced carotenogenesis. The CarD N-terminal domain, which defines the large CarD_CdnL_TRCF protein family, binds to CarG, a zinc-associated protein that does not bind DNA. The CarD C-terminal domain resembles eukaryotic high-mobility-group A (HMGA) proteins, and its DNA binding AT hooks specifically recognize the minor groove of appropriately spaced AT-rich tracts. Here, we investigate the determinants of the only known CarD binding site, the one crucial in CarD-CarG regulation of the promoter of the carQRS operon (P_QRS), a light-inducible promoter dependent on the extracytoplasmic function (ECF) σ factor CarQ. In vitro, mutating either of the 3-bp AT tracts of this CarD recognition site (TTTCCAGAGCTTT) impaired DNA binding, shifting the AT tracts relative to P_QRS had no effect or marginally lowered DNA binding, and replacing the native site by the HMGA1a binding one at the human beta interferon promoter (with longer AT tracts) markedly enhanced DNA binding. In vivo, however, all of these changes deterred P_QRS activation in wild-type M. xanthus, as well as in a strain with the CarD-CarG pair replaced by the Anaeromyxobacter dehalogenans CarD-CarG (CarD_Ad-CarG_Ad). CarD_Ad-CarG_Ad is functionally equivalent to CarD-CarG despite the lower DNA binding affinity in vitro of CarD_Ad, whose C-terminal domain resembles histone H1 rather than HMGA. We show that CarD physically associates with RNA polymerase (RNAP) specifically via interactions with the RNAP β subunit. Our findings suggest that CarD regulates a light-inducible, ECF σ-dependent promoter by coupling RNAP recruitment and binding to a specific DNA site optimized for affinity and position.     

Nuclear factors interact with conserved A/T-rich elements upstream of a nodule-enhanced glutamine synthetase gene from French bean.

The gln-gamma gene, encoding the gamma subunit of glutamine synthetase in French bean (Phaseolus vulgaris), is strongly induced during nodule development. We have determined the nucleotide sequence of a 1.3-kilobase region at its 5' end and have identified several sequences common to the promoter regions of late nodulin genes from other legume species. The 5'-flanking region was analyzed for sequence-specific interactions with nuclear factors from French bean. A factor from nodules (PNF-1) was identified that binds to multiple sites between -860 and -154, and a related but distinct factor (PRF-1) was detected in extracts from uninfected roots. PNF-1 and PRF-1 bound strongly to a synthetic oligonucleotide containing the sequence of an A/T-rich 21-base pair imperfect repeat found at positions -516 and -466. The same factors also had a high affinity for a protein binding site from a soybean leghemoglobin gene and appeared to be closely related to the soybean nodule factor NAT2, which binds to A/T-rich sequences in the lbc3 and nodulin 23 genes [Jacobsen et al. (1990). Plant Cell 2, 85-94]. Comparison of NAT2/PNF-1 binding sites from a variety of nodulin genes revealed the conservation of the short consensus core motif TATTTWAT, and evidence was obtained that this sequence is important for protein recognition. Cross-recognition by PNF-1 of a protein binding site in a soybean seed protein gene points to the existence of a ubiquitous family of factors with related binding affinities. Our data suggest that PNF-1 and PRF-1 belong to an evolutionarily conserved group of nuclear factors that interact with specific A/T-rich sequences in a diverse set of plant genes. We consider the possible role of these factors in coregulating the expression of gln-gamma and other late nodulin genes.     

Sequence-specific binding of HMG-I(Y) to the proximal promoter of the gp91-phox gene.

Screening of a cDNA expression library with a CCAAT-box element derived from the myelomonocyte-specific gp91-phox promoter resulted in the isolation of three independent HMG-I(Y) cDNA clones. Filter binding competition studies reveal that HMG-Y binds to this promoter element in a sequence-specific manner and exhibits a gradient of binding affinities for various A/T-rich sequences. Two adjacent A/T-rich regions within the gp91-phox promoter CCAAT-box element are required for maximal binding. In addition, competition experiments demonstrate that the binding affinity of HMG-Y is influenced by sequences that flank A/T-rich core binding sites.