A new member of the Balbiani ring multigene family in the dipteran Chironomus tentans consists of a single-copy version of a unit repeated in other gene family members

The known Balbiani ring (BR) multigene family members in the dipteran Chironomus tentans encode salivary gland secretory proteins in the size range between 38 and 1,000 kDa. The proteins interact to form protein fibers used by the aquatic larvae to spin feeding and protective larval tubes or pupation tubes. Here, we describe a new BR multigene family member, the spl7 gene, which codes for an 89-amino-acid-long protein with a relative mobility of 17k. The gene has a high content of charged amino acid residues and consists of two structurally different halves. Five regularly spaced cysteine codons are present in the 5 half while the 3 half contains five proline codons. These two different halves exhibit similarities to the C and SR regions, respectively, which form the tandemly repeated units in the about 40-kb-long BR genes and which also, in different versions, are the building blocks of all genes in the BR multigene family.In this multigene family, encoding interacting structural proteins, the long BR genes with their 125–150 tandemly arranged repeat units as well as the short sp17 gene with its single-copy version of such a repeat unit, have therefore evolved from a common ancestor.Correspondence to: L. Wieslander     

Conserved and nonconserved structures in the secretory proteins encoded in the Balbiani ring genes ofChironomus tentans

Summary The large, repetitive Balbiani ring (BR) genes, BR 1, 2, and 6, inChironomus tentans originated from a short ancestral sequence and have all evolved according to analogous amplification schemes. We analyzed the structures of the BR-encoded secretory proteins and defined the parts that have been conserved during the evolutionary process. The BR products show striking similarities, with the BR 1 and BR 2 products being more similar to each other than to the BR 6 product. In the constant (C) region of the repeat units, 7 of the 30 amino acid residues are strictly conserved; 4 of these are the cysteine residues. The subrepeat (SR) regions of all the BR products are dominated by repeated tripeptide elements rich in proline and charged amino acid residues. Most of the amino acid replacements in both regions are conservative. Secondary structure predictions suggested that the C regions of the BR 1 and BR2 products have several elements of secondary structure: an -helix, a -strand, and one or two reverse turns, as in globular structures. The prediction for the C region of the BR 6 product is similar but lacks a -strand. The predictions for the intervening SR regions appear less conclusive, but are clearly different from those for the C regions, and suggest regular structures not differing in their conformational elements. The SR regions evolved from an ancestor sequence similar to the C region; thus, the BR products seem to represent an example of evolution from one structure to two differently folded products. It is proposed that the alignment and polymerization of the long BR proteins could be promoted by the repetitive structure of the molecules, due to the possibility of forming disulfide bridges between half-cystine residues and electrostatic interactions between the charged residues of the SR regions. The divergence among the BR products is discussed in relation to possible functional differences among the members of the BR gene family.     

Conserved and variable repeat structures in the Balbiani ring gene family in Chironomus tentans

Summary The four Balbiani ring (BR) genes, BR1, BR2.1, BR2.2, and BR6 in the midge Chironomus tentans constitute a gene family encoding secretory proteins with molecular weights of approximately 10⁶ daltons. The major part of each gene is known to consist of tandemly organized composite repeat units resulting in a hierarchic repeat arrangement.Here, we present the sequence organization of the 5 part of the BR2.2 and BR6 genes and describe the entire transcribed part of the two genes. As the BR1 and BR2.1 genes were also fully characterized recently, this allows the comparison of all genes in the BR gene family.All four genes share the same exon-intron structure and have evolved by gene duplications starting from a common ancestor, having the same overall organization as the BR genes of today.The genes encode proteins that have an approximately 10,000-amino acid residue extended central domain, flanked by a highly charged, 200-residue amino-terminal domain and a globular 110-residue carboxy-terminal domain. Exons 1–3 and the beginning of exon 4 encode the amino-terminal domain, which throughout contains many regions built from short repeats. These repeats are often degenerate as to repeat unit and sequence and are present in different numbers between the genes. In several instances these repeat structures, however, are conserved at the protein level where they form positively or negatively charged regions.Each BR gene has a 26–38-kb-long exon 4, which consists of an array of 125–150 repeat units and encodes the central domain. The number of repeat units appears to be largely preserved by selection and all repeat units in the array are very efficiently homogenized. Occasionally variant repeats have been introduced, presumably from another BR gene by gene conversion, and spread within the array.Introns 1–3 at the 5 end of the genes have diverged extensively in sequence and length between the genes. In contrast, intron 4 at the 3 end is virtually identical between three of the four genes, suggesting that gene conversion homogenizes the 3 ends of the genes, but not the 5 ends. Offprint requests to: L. Wieslander     

Balbiani ring DNA: Sequence comparisons and evolutionary history of a family of hierarchically repetitive protein-coding genes

Summary All known types of Balbiani ring (BR) gene consist of multiple, tandemly arranged, ca. 180 to 300-bp repeat units that can be divided into a constant region and a subrepeat region. The latter region includes short tandem subrepeats (SRs). Comparison of all available BR sequences using computer methods has enabled us (a) to define more precisely the constant and subrepeat regions, (b) to infer the evolutionary relationships among the various types of BR repeats, (c) to derive a consensus approximation of an ancestral sequence from a small segment of which the highly diverse present-day SRs may have originated, and (d) to detect an underlying substructure in the constant region, evident in the consensus but not in the present-day sequences and possibly corresponding to an original 39-bp DNA segment from which the extant, giant BR sequences may have evolved. We discuss the processes of reduplication, diversification, and homogenization within the hierarchically repetitive BR sequences as examples of how a simple DNA element may evolve into a diverse family of large, protein-coding genes.     

The Chironomus tentans genome sequence and the organization of the Balbiani ring genes

Background

The polytene nuclei of the dipteran Chironomus tentans (Ch. tentans) with their Balbiani ring (BR) genes constitute an exceptional model system for studies of the expression of endogenous eukaryotic genes. Here, we report the first draft genome of Ch. tentans and characterize its gene expression machineries and genomic architecture of the BR genes.

Results

The genome of Ch. tentans is approximately 200 Mb in size, and has a low GC content (31%) and a low repeat fraction (15%) compared to other Dipteran species. Phylogenetic inference revealed that Ch. tentans is a sister clade to mosquitoes, with a split 150–250 million years ago. To characterize the Ch. tentans gene expression machineries, we identified potential orthologus sequences to more than 600 Drosophila melanogaster (D. melanogaster) proteins involved in the expression of protein-coding genes. We report novel data on the organization of the BR gene loci, including a novel putative BR gene, and we present a model for the organization of chromatin bundles in the BR2 puff based on genic and intergenic in situ hybridizations.

Conclusions

We show that the molecular machineries operating in gene expression are largely conserved between Ch. tentans and D. melanogaster, and we provide enhanced insight into the organization and expression of the BR genes. Our data strengthen the generality of the BR genes as a unique model system and provide essential background for in-depth studies of the biogenesis of messenger ribonucleoprotein complexes.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-819) contains supplementary material, which is available to authorized users.     

Cerato-platanin, the first member of a new fungal protein family

The ascomcete Ceratocystis fimbriata, the causal agent of “canker stain disease,” secretes a protein of 12.4 kDa that elicits phytoalexin synthesis and plant cell death. This protein, named cerato-platanin (CP), is also located in the cell walls of ascospores, hyphae, and conidia; it contains four cysteines (S-S bridged) and is moderately hydrophobic. The cp gene consists of a single exon and has 42 bp codifying for a signal peptide of 14 residues. The recombinant protein was obtained by cloning the cp gene of the mature protein in Escherichia coli (BL21), and a refolding step was needed to achieve the native active form. In the European Molecular Biology data bank, CP is reported as the first member of the CP family; this is the first example of an set of secreted fungal proteins whose primary structure is very similar. Nonetheless, the data also revealed some structural and functional features that make CP simlar to proteins of the hydrophobin family.     

Mouse sperm exhibit chemotaxis to allurin, a truncated member of the cysteine-rich secretory protein family

Allurin, a 21 kDa protein isolated from egg jelly of the frog Xenopus laevis, has previously been demonstrated to attract frog sperm in two-chamber and microscopic assays. cDNA cloning and sequencing has shown that allurin is a truncated member of the Cysteine-Rich Secretory Protein (CRISP) family, whose members include mammalian sperm-binding proteins that have been postulated to play roles in spermatogenesis, sperm capacitation and sperm–egg binding in mammals. Here, we show that allurin is a chemoattractant for mouse sperm, as determined by a 2.5-fold stimulation of sperm passage across a porous membrane and by analysis of sperm trajectories within an allurin gradient as observed by time-lapse microscopy. Chemotaxis was accompanied by an overall change in trajectory from circular to linear thereby increasing sperm movement along the gradient axis. Allurin did not increase sperm velocity although it did produce a modest increase in flagellar beat frequency. Oregon Green 488-conjugated allurin was observed to bind to the sub-equatorial region of the mouse sperm head and to the midpiece of the flagellum. These findings demonstrate that sperm have retained the ability to bind and respond to truncated Crisp proteins over 300 million years of vertebrate evolution.     

Balbiani ring 6 gene in Chironomus tentans: a diverged member of the Balbiani ring gene family

We describe the internal organization of a large part of the Balbiani ring (BR) 6 gene in Chironomus tentans. The BR6 gene is a diverged member of the BR gene family. It displays the characteristic hierarchic organization of repetitive sequences, but in the constant region of the repeat units the overall sequence homology is only 49% when compared to other BR genes. All four cysteines are among the few amino acids conserved in the constant region. In the subrepeat region the central part is built from a repeated tripeptide, Pro-Glu--Arg+. A similar charge distribution adjacent to prolines is found in other BR gene subrepeat regions, most pronouncedly in the BR2-encoded protein. These conserved properties of the BR gene products are relevant to the issue how the various BR gene products interact to form a supramolecular structure, the larval tube, and how functional demands influence the evolution of a eucaryotic gene family.     

The bromodomain protein LEX-1 acts with TAM-1 to modulate gene expression in C. elegans

In many organisms, repetitive DNA serves as a trigger for gene silencing. However, some gene expression is observed from repetitive genomic regions such as heterochromatin, suggesting mechanisms exist to modulate the silencing effects. From a genetic screen in C. elegans, we have identified mutations in two genes important for expression of repetitive sequences: lex-1 and tam-1. Here we show that lex-1 encodes a protein containing an ATPase domain and a bromodomain. LEX-1 is similar to the yeast Yta7 protein, which maintains boundaries between silenced and active chromatin. tam-1 has previously been shown to encode a RING finger/B-box protein that modulates gene expression from repetitive DNA. We find that lex-1, like tam-1, acts as a class B synthetic multivulva (synMuv) gene. However, since lex-1 and tam-1 mutants have normal P granule localization, it suggests they act through a mechanism distinct from other class B synMuvs. We observe intragenic (interallelic) complementation with lex-1 and a genetic interaction between lex-1 and tam-1, data consistent with the idea that the gene products function in the same biological process, perhaps as part of a protein complex. We propose that LEX-1 and TAM-1 function together to influence chromatin structure and to promote expression from repetitive sequences. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A staphylococcal multidrug resistance gene product is a member of a new protein family.

The complete nucleotide sequence (321 bp) of smr (staphylococcal multidrug resistance), a gene coding for efflux-mediated multidrug resistance of Staphylococcus aureus, was determined by using two different plasmids as DNA templates. The smr gene product (identical to products of ebr and qacC/D genes) was shown to be homologous to a new family of small membrane proteins found in Escherichia coli, Pseudomonas aeruginosa, Agrobacterium tumefaciens, and Proteus vulgaris. The smr gene was subcloned and expressed in S. aureus and E. coli and its ability to confer the multidrug resistant phenotype was demonstrated for two different lipophilic cation classes: phosphonium derivatives and quarternary amines. Expression of smr gene leads to the efflux of tetraphenylphosphonium and to a net decrease in the uptake of lipophilic cations. The deduced polypeptide sequence (107 amino acid residues, 11,665 kDa) has 46% hydrophobic residues (Phe, Ile, Leu, and Val) and 20% hydroxylic residues (Ser and Thr). Four transmembrane segments are predicted for smr gene product. Of the charged amino acid residues, only Glu 13 is located in a transmembrane segment. This Glu 13 is conserved in all members of the family of small membrane proteins. We propose a mechanism whereby exchange of protons at the Glu 13 is a key in the efflux of the lipophilic cation. This mechanism includes the idea that protons are transported to the Glu 13 via an appropriate chain of hydroxylic residues in the transmembrane segments of Smr.     

A gene encoding a novel isoform of the PR-1 protein family from tomato is induced upon viroid infection

A Lycopersicon esculentum cDNA clone encoding an acidic-type pathogenesis-related protein (PR-lal) was isolated, sequenced and characterized. It contains an open reading frame of 175 amino acids and the mature protein, after cleavage of the 21 amino acid signals peptide, has a pl of 5.24. The protein shows highest homology (75% identity) with the basic pathogenesis-related prb-lb protein from tobacco. The PR-lal gene shows constitutive expression in roots from tomato plants. It is expressed in leaves and stems upon viroid infection, and appears to be induced by ethylene. Comparative studies of this gene and a related basic isoform of PR-1 indicate that the expression of these two members of the PR-1 gene family in tomato may be differentially regulated upon viroid infection.The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X71592.     

Expression pattern and gene characterization of asporin. a newly discovered member of the leucine-rich repeat protein family

We have discovered a new member of the class I small leucine-rich repeat proteoglycan (SLRP) family which is distinct from the other class I SLRPs since it possesses a unique stretch of aspartate residues at its N terminus. For this reason, we called the molecule asporin. The deduced amino acid sequence is about 50% identical (and 70% similar) to decorin and biglycan. However, asporin does not contain a serine/glycine dipeptide sequence required for the assembly of O-linked glycosaminoglycans and is probably not a proteoglycan. The tissue expression of asporin partially overlaps with the expression of decorin and biglycan. During mouse embryonic development, asporin mRNA expression was detected primarily in the skeleton and other specialized connective tissues; very little asporin message was detected in the major parenchymal organs. The mouse asporin gene structure is similar to that of biglycan and decorin with 8 exons. The asporin gene is localized to human chromosome 9q22-9q21.3 where asporin is part of a SLRP gene cluster that includes extracellular matrix protein 2, osteoadherin, and osteoglycin. Further analysis shows that, with the exception of biglycan, all known SLRP genes reside in three gene clusters.     

Evolution of the interleukin-1 gene family in mammals

The phylogeny of interleukin-1 family genes shows that human interleukin-1 (IL-1) is more closely related to IL-1 of the bovine than to IL-1 of the mouse, whereas human interleukin-1 (IL-1) is more closely related to IL-1 of the mouse than to IL-1 of the bovine. The IL-1 receptor antagonist (IL-1) shows homology to the C-terminal region of both IL-1 and IL-1. In the C-terminal region, the IL-1 genes of human and mouse have diverged more from each other at nonsynonymous sites than have either IL-1 or IL-1; because the same pattern is not seen at synonymous sites, it must be due not to a difference in mutation rate but rather to a greater degree of functional constraint on this region in the IL-1 and IL-1 proteins than in the IL-1 protein. But synonymous sites in IL-1 of mouse have evolved more rapidly than in IL-1 of human, indicating a higher rate of mutation in the former gene. In the N-terminal region of the protein, nonsynonymous sites have evolved at similar rates in IL-1 and IL-1. The first exon of the IL-1 gene, which encodes the leader peptide, shows evidence of homology with the first exon of IL-1, which is not translated. Thus, it seems likely that IL-1 evolved by duplication of an IL-1 gene and loss of expression of exons 2–4. Correspondence to: A.L. Hughes     

The characterization of the soybean polygalacturonase-inhibiting proteins (Pgip) gene family reveals that a single member is responsible for the activity detected in soybean tissues

Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins that inhibit fungal endopolygalacturonases (PGs). They are encoded by multigene families whose members show functional redundancy and subfunctionalization for recognition of fungal PGs. In order to expand the information on the structure and functional features of legume PGIP, we have isolated and characterized four members of the soybean Pgip gene family and determined the properties of the encoded protein products. Sequence analysis showed that these genes form two clusters: one cluster of about 5 kbp containing Gmpgip1 and Gmpgip2, and the other containing Gmpgip3 and Gmpgip4 within a 60 kb fragment of a separate BAC clone. Sequence diversification of the four members resides mainly in the xxLxLxx region that includes residues forming the β-sheet B1. When compared with other legume Pgip genes, Gmpgip3 groups with the bean genes Pvpgip1 and Pvpgip2, suggesting that these genes are closer to the ancestral gene. At the protein level, only GmPGIP3 shows the capability to inhibit fungal PGs. The spectrum of inhibition of GmPGIP3 against eight different fungal PGs mirrors that of the PGIP purified from soybean tissues and is similar to that of the bean PvPGIP2, one of the most efficient inhibitors so far characterized. We also report that the four Gmpgip genes are differentially regulated after wounding or during infection with the fungal pathogen Sclerotinia sclerotiorum. Following fungal infection Gmpgip3 is up regulated promptly, while Gmpgip2 is delayed.