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     

Evolutionary conservation of the 3′ ends of members of a family of giant secretory protein genes inChironomus pallidivittatus

Summary Two cDNA clones representing the 3-end regions of BR1 and BR2 75S mRNA were obtained fromChironomus pallidivittatus. The regular structure characterizing the core of these genes, consisting of tandemly arranged repeat units, changes into a more irregular structure toward the 3 end. Distal to a standard type of repeat unit with a characteristic excess of positive charges, a new type of repeat with a high, negative charge density is interspersed among parts of the standard unit. The last 111 amino acids before the stop codon represent a unique region distinctly different in amino acid composition from upstream regions, and include two partially homologous hydrophobic regions. Sequence comparison of 3-end regions from clones representing BR1 and BR2 genes indicates striking sequence conservation in the unique part of the region. Analysis of the level of silent site divergence shows that the homology increases in the 3 direction up to the polyadenylation site. That the unique region is retained as a part of the secreted protein is shown by Western blotting.     

Structural implications of primary sequences from a family of Balbiani ring-encoded proteins inChironomus

Summary DNA sequencing has revealed an internal, tandemly repetitive structure in the family of giant polypeptides encoded by three types of Balbiani ring (BR) genes, in three different species ofChironomus. Each major BR repeat can be subdivided into two halves: a region consisting of short subrepeats and a more constant region that lacks obvious subrepeats. Comparative predictions of secondary structure indicate that an -helical segment is consistently present in the amino-terminal half of the constant region in all known BR proteins. Comparative predictions, coupled with consideration of the known phosphorylation of serine and threonine residues in BR proteins, suggest that the -helical structure may also extend into the carboxy-terminal half of the constant region, possibly interrupted by -turn(s). However, it is also possible that the structure is variable, and that a -strand is present in that half in some cases. All of the constant regions conserve one methionine and one phenylalanine residue, as well as all four cysteines; these residues presumably play roles in the packing or cross-linking of aligned constant regions. The structure of the subrepeat region is not clear, but the prevalence of a tripeptide pattern (basic-proline-acidic) suggests some type of structural regularity, possibly an extended helix. The possible significance of these conserved molecular features is discussed in the context of how they may serve the elasticity, insolubility, and hydrophilicity of the fibrils and threads formed by the BR polypeptides.     

Differential display-mediated rapid identification of different members of a multigene family, HSP16.9 in wheat

Isolation of cDNAs encoding individual members of a gene family is essential for assessing their role in a biological phenomenon. However, this process is often laborious and slow due to highly conserved protein-coding region that interferes with the isolation of the individual members. Identification of gene-specific probes from 3 non-coding regions of different members can assist in the fast retrieval and characterization of individual members of a multigene family. We used the recent technique of differential display for the same purpose. As an example of a multigene family in plants, we selected a heat shock protein gene family, HSP16.9 from wheat, with estimated 12 members. We modified the original differential display technique for selective amplification of the 3 non-coding regions of different wheat HSP16.9 genes by replacing the random 10-mer in the original method with a conserved HSP16.9 gene family-specific primer. Sixteen cDNA fragments from these experiments were sequenced and they represent 8 different members of a 12 member gene family. Our succes can be attributed to shorter 3 non-coding regions that are typical of higher-plant genes and use of highly conserved gene family-specific primer in these experiments. This modified differential display technique can be of general application to other plant systems where cloning of the different members of a gene family is desired.     

The 3′ ends of two genes in the Balbiani ring c locus ofChironomus thummi

Summary The 3-end sequences of two nonallelic genes derived from the Balbiani ring c (BRc) locus ofChironomus thummi are described. Only one of the genes appears to be transcribed abundantly in normal late larval salivary glands. The two sequences are highly similar, even in the 3 untranslated regions, but sharply diverge beyond the polyadenylation site. Together with evidence from the 3 ends of BR1 and BR2 genes ofC. pallidivittatus andC. tentans, independently characterized by others, this result suggests the existence of a sequence-homogenization mechanism that operates across the 3 ends of all BR genes characterized to date. The 3-terminal coding region of each BRc gene is divided into two portions by a short intron. The upstream portion is homologous to and continuous with the tandem repeats that make up the internal core of each BR gene; however, that portion is variant in sequence relative to the core, and apparently is not subject to the homogenization process that operates on the core repeats. The portion downstream of the intron encodes a unique, 111-residue polypeptide highly different from the rest of the BRc product. The evolution of the various segments of the BRc genes is discussed.     

Thecab-m7 gene: a light-inducible,mesophyll-specific gene of maize

Southern blot analysis has revealed the existence in maize of perhaps 12 members of the nuclearcab multigene family encoding the chlorophylla- andb-binding proteins of the photosystem II light-harvesting complex. Hybridization with 3 probes derived from unsequenced cDNA clones showed that six members of this family differ from one another with respect to expression in mesophyll and/or bundle sheath cells and regulation by light. An additional member of this family, designatedcab-m7, that encodes a 28 kDa primary translation product has now been identified. It has been cloned from a maize genomic library and sequenced to begin to define the bases for differences in the expression of these genes. Thiscab gene is shown to be strongly preferentially expressed in the mesophyll (vs. bundle sheath) cells of maize. Furthermore, the gene is photo-responsive; although small amounts ofcab-m7 mRNA are present in etiolated leaves, the mRNA pool is 8-fold larger after six hours of illumination. DNA sequences upstream of thecab-m7 gene resemble those found in the 5-flanking regions of some other plant genes.     

Identification and chromosomal location of four subfamilies of the rubisco small subunit genes in common wheat

Summary Three different 3 noncoding sequences of wheat rubisco small subunit (SSU) genes (RbcS) were used as probes to identify the gene members of different RbcS subfamilies in the common wheat cultivar Chinese Spring (CS). All genes of the wheat RbcS multigene family were previously assigned to the long arm of homoeologous group 5 and to the short arm of homoeologous group 2 chromosomes of cv CS. Extracted DNA from various aneuploids of these homoeologous groups was digested with four restriction enzymes and hybridized with three different 3 noncoding sequences of wheat SSU clones. All RbcS genes located on the long arm of homoeologous group 5 chromosomes were found to comprise a single subfamily, while those located on the short arm of group 2 comprised three subfamilies. Each of the ancestral diploid genomes A, B, and D has at least one representative gene in each subfamily, suggesting that the divergence into subfamilies preceded the differentiation into species. This divergence of the RbcS genes, which is presumably accompanied by a similar divergence in the 5 region, may lead to differential expression of various subfamilies in different tissues and in different developmental stages, in response to different environmental conditions. Moreover, members of one subfamily that belong to different genomes may have diverged also in the coding sequence and, consequently, code for distinguishable SSU. It is assumed that such utilization of the RbcS multigene family increases the adaptability and phenotypic plasticity of common wheat over its diploid progenitors.     

Tissue-specific and developmental regulation of a gene encoding a low molecular weight sulfur-rich protein in soybean seeds

A gene corresponding to a cDNA clone, SE60, encoding a low molecular weight sulfur-rich protein in soybean seeds was isolated from a soybean genomic library and characterized at the nucleotide level. The SE60 gene is interrupted by an intervening sequence of 694 by in size. The 5 flanking region of the gene contained various regulatory sequences such as the RY repeat and CACA elements found in other seed protein genes of legumes. The SE60 gene encoded a pre-protein of 75 amino acids, having a signal sequence of 28 amino acids at the N-terminus. The mature protein of 47 amino acids was basic and cysteine-rich. Northern blot analysis suggested that the SE60 gene is expressed in a tissue-specific and developmentally regulated manner during soybean seed development. The SE60 genes form a small multigene family composed of about four members in the soybean genome.     

No evidence for linkage of long QT syndrome and chromosome 11p15.5 markers in a Chinese family: evidence for genetic heterogeneity

Recently the defective gene locus in seven Caucasian families with the Romano-Ward form of long QT syndrome (LQT) has been mapped to chromosome 11p. To understand the molecular basis of LQT in Chinese, a three-generation family was investigated. Fourteen family members were studied and five individuals were diagnosed to be affected, according to electrocardiographic criteria. Two genomic DNA probes (c-Ha-ras-3-HVR and insulin-5-HVR) and one tetranucleotide repeat polymorphism (THZ) derived from chromosome 11p15.5 loci and previously demonstrated to be closely linked to LQT were used as probes to analyze this family. A lod score of less than -2 was noted for all three polymorphisms. Our data show that there was no evidence of linkage between these three loci and the gene for LQT in this studied family. We believe that this result provides additional evidence for genetic heterogeneity of LQT.     

Characterization of the supervirulent virG gene of the Agrobacterium tumefaciens plasmid pTiBo542.

Summary The virG gene of the Agrobacterium tumefaciens Ti plasmid pTiBo542 has previously been reported to elicit stronger vir gene expression than its counterpart in the pTiA6 plasmid, a property we call the superactivator phenotype. The DNA sequence of the pTiBo542 virG gene was determined and compared to that of the pTiA6 gene. The DNA sequences of these genes differ at 16 positions: two differences are in the promoter regions, 12 are in the coding regions, and two are in the 3 untranslated regions. The 3 end of the pTiA6 virG gene also contains a probable insertion sequence that is not found downstream of the pTiBo542 gene. The base pair differences in the two coding regions result in only two amino acid differences, both in the amino-terminal halves of the proteins. Five hybrid virG genes were constructed and used to activate the expression of a virB::lacZ gene fusion. Differences in the coding regions of these genes accounted for most of the superactivator phenotype, while differences at the promoter and 3 untranslated regions also contributed. These findings suggest that the properties of these VirG proteins and their quantities are important for vir gene induction, and also suggest a long-term selective pressure for mutations contributing to differences between these two genes.     

Integrons: Novel DNA elements which capture genes by site-specific recombination

Integrons are unusual DNA elements which include a gene encoding a site-specific DNA recombinase, a DNA integrase, and an adjacent site at which a wide variety of antibiotic resistance and other genes are found as inserts. One or more genes can be found in the insert region, but each gene is part of an independent gene cassette. The inserted genes are expressed from a promoter in the conserved sequences located 5 to the genes, and integrons are thus natural expression vectors. A model for gene insertion in which circular gene cassettes are inserted individually via a single site-specific recombination event has been proposed and verified experimentally. The gene cassettes include a gene coding region and, at the 3 end of the gene an imperfect inverted repeat, a 59-base element. The 59-base elements are a diverse family of elements which function as sites recognized by the DNA integrase. Site-specific insertion of individual genes thus represents a further mechanism which contributes to the evolution of the genomes of Gram-negative bacteria and their plasmids and transposons.Members of the most studied class of integrons, which include thesulI gene in the conserved sequences, are believed to be mobile DNA elements on the basis that they are found in many independent locations, and a discrete boundary is found at the outer end of the 5-conserved segment. However, the length of the 3-conserved segment is variable in the integrons examined to date, and it is likely that this variability has arisen as the result of insertion and deletion events. Though the true extent of the 3-conserved segment remains to be determined, it seems likely that these integrons are mobile DNA elements. The second known class of integrons comprises members of the Tn7 transposon family.     

Characterization of a Gy4 glycinin gene from soybean Glycine max cv. Forrest

The glycinin gene family encoding the glycinin subunits in soybean plants is composed of at least five gene members. A genomic clone S312 containing the Gy4 gene from a genomic library of cv. Forrest was isolated and partially characterized. The organization of this gene was found to be similar to that of a null allele from cv. Raiden, but different from the Gy4 gene from cv. Dare. The complete nucleotide sequence of this gene has been determined. It is 2599 bp long consisting of four exons and three introns. Comparing the DNA sequences between this gene and the gene from Dare and a null allele from Raiden, the difference found in the coding region was 5-GCAGTGCAAG-3 (nt 824 to 833) in the former case versus 5-TGGAGTTGCAATT-3 (nt 1314 to 1326) in the latter case in the exon 2 domain, resulting in three amino acid differences and one amino acid absence. Some other differences were also found in the non-coding region. The coding sequence and 5-flanking region of the Gy4 gene, when compared with that of other legumin genes as well as group 1 glycinin subunit genes, revealed some interesting features: (1) a transposable element-like sequence was found in the hypervariable region (HVR) of the exon 3 domain, which was lacking in the legumin and the glycinin group 1 genes; (2) in the 5-flanking region from nt –145 to –1, two high-homology sequences were found: one from nt –141 to nt –132, the other from nt –118 to nt –92 which includes the legumin box and the RY repeat element.     

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.     

The organization of repetitive sequences in the albumin and alpha-fetoprotein gene loci in the rat

Summary The distribution of middle repetitive sequences in the genic and extragenic regions of the rat albumin and -fetoprotein genes was analyzed. Their presence was determined by probing Southern blots of restriction fragments of albumin and -fetoprotein genomic subclones with ³²P-labeled total rat DNA. Repetitive sequences were detected in both genes. They were classified as weak, moderate and intense hybridizing elements according to the intensity of hybridization. Weak repetitive sequences were characterized as dG·dT repeats by using ³²P-labeled poly-(dG·dT)(dC·dA) oligomer probe. They occurred in 5 and 3 extragenic regions of the two genes and in introns 4 and 5 of the albumin gene. The moderate repetitive sequence present in intron 6 of the albumin gene was identified as the rat SINES element, 4D12. The intense repetitive sequence, localized in the 3 non-coding region of the albumin gene, corresponded to the terminal segment of a rat high repeat long interspersed DNA family, L1Rn. 4D12 and L1Rn sequences were also scattered throughout the -fetoprotein locus as moderate and intense repetitive elements, respectively, but their distribution was different from that of the albumin genomic region. These results indicate that repetitive sequences invaded the two loci in a non-conservative manner.     

Subrepeats within the BR1β repeat unit inChironomus pallidivittatus can be classified into different types depending on codon usage

Summary A new type of repeat unit was isolated from Balbiani ring 1 ofChironomus pallidivittatus and designated, BR1 repeat. It consists of a constant and a subrepeated part, like previously described units belonging to the core blocks of the BR genes. The subrepeated part contains 10-codon subrepeats with an arrangement similar to the subrepeats of the previously described BR2 gene. The present unit differs from earlier reported core units firstly in a much lower number of copies (about 15) per genome, which are tandemly arranged. Secondly, the number of subrepeats per BR1 repeat unit can show great variations. On the basis of the pattern of codon usage, three types of subrepeats can be distinguished. One type lies 5-proximal in the subrepeat array and consists of variable numbers of subrepeats almost identical at the nucleotide level. The last complete subrepeat represents another type, with consistent differences in codon usage as compared to subrepeats of the proximal type. Finally, there is an intermediate type represented by the subrepeat preceding the distal one. Here, codon characteristics from proximal and distal subrepeats are mixed in a patchy and irregular way. The evolution of the arrays can be understood either as being the result of subrepeat formation in two steps (occurring before and after amplification of whole repeat units) or as the result of a continuous process in which there is evidence for participation of gene conversion.     

Characterization of a pollen-specific gene family from Brassica napus which is activated during early microspore development

In this paper we describe the isolation and characterization of a genomic clone (Bp4) from Brassica napus which contains three members of a pollen-specific multigene family. This family is composed of 10 to 15 closely related genes which are expressed in early stages of microspore development. The complete nucleotide sequence of the clone Bp4 and of three homologous cDNA clones is reported. One of the genes (Bp4B) contained in the genomic clone is believed to be non-functional because of sequence rearrangements in its 5 region and intron splicing sites. The remaining genes (Bp4A and Bp4C), as well as the cDNA clones, appear to code for small proteins of unique structure. Three different types of proteins can be predicted as a result of the deletion of carboxy or amino terminal portions of a conserved core protein. These proteins all share a common alternation of hydrophobic and hydrophilic domains. A fragment of the genomic clone containing the gene Bp4A, as well as the non-functional gene Bp4B, was introduced into tobacco plants via Agrobacterium-mediated transformation. The functional gene Bp4A is expressed in transgenic tobacco plants and shows spatial and temporal regulation consistent with the expression patterns seen in Brassica napus.     

The adult α-globin locus of old world monkeys: An abrupt breakdown of sequence similarity to human is defined by an Alu family repeat insertion site

Summary The haploid genomes of all known primates have two or more adult -globin genes contained within tandemly arranged duplication units. Although the tandem duplication event generating these -globin loci is believed to occur prior to the divergence of primates, a number of length polymorphisms exist within the loci among different primate species. In order to understand the molecular basis of these length polymorphisms, we have cloned and determined the nucleotide sequence of a major portion of the rhesus monkey adult -globin locus. Sequence comparison to human suggests that the length difference between the adult -globin loci of human and Old World monkey is the result of one or more DNA recombination processes, all of which appeared to be related to the transposition of Alu family repeats. First, the finding of a monomeric Alu family repeat at the junction between nonhomology block I and homology block Y of the 2 genecontaining unit in rhesus macaque suggests that the dimeric Alu family repeat, Alu 3, at the orthologous position in human was generated by insertion of a monomeric Alu family repeat into the 3 end of another preexisting Alu family repeat. Second, two Alu family repeats, Alu 1 and Alu 2, exist in human at the 3 end of each of the two X homology blocks, respectively. However, this pair of paralogous Alu family repeats is absent at the corresponding positions in rhesus macaques. This raises interesting questions regarding the evolutionary origin of Alu 1 and Alu 2. Finally, DNA sequences immediately downstream from the insertion site of Alu 2 are completely different between human and rhesus macaque. This last event is similar to DNA rearrangements occurring nearby transposable element(s) in the chromosomes of bacteria, yeast, and plant cells. Its possible role in accelerating the genomic evolution of noncoding or spacer DNA is discussed.     

Genes encoding light-harvesting and reaction center proteins from Chromatium vinosum

Sequencing of a 3.4 kb DNA fragment isolated from the photosynthetic purple sulfur bacterium Chromatium vinosum and of PCR products has resulted in identification of the Chr. vinosum pufL, pufM, and pufC genes, reading from the 5 to the 3 direction, and coding, respectively, for the L, M and cytochrome c subunits of the reaction center of this bacterium. Other PCR products have been used to obtain complete sequences for the pufB and pufA genes, located immediately upstream from pufL and encoding the apoproteins of two Chr. vinosum light- harvesting proteins. The 3-portion of the bchZ gene, a gene that codes for a protein involved in the biosynthesis of bacteriochlorophyll, has been located immediately upstream from pufB. A second pufB gene, pufB2, has been located downstream from pufC, as has the 5-portion of a second pufA gene, pufA2. The location of a second set of pufB and pufA genes, encoding light- harvesting proteins, downstream from pufC has not previously been reported for any photosynthetic bacterium. Translation of the gene sequences encoding these Chr. vinosum light-harvesting proteins reveals both similarities to and differences from the amino acid sequences, obtained from direct sequencing of the apoproteins, previously reported for Chr. vinosum light-harvesting proteins. Translation of these gene sequences, and of those for pufL, pufM and pufC, revealed significant homology, at the amino acid level, to the corresponding peptides of photosynthetic purple non-sulfur bacteria.     

Histone H4 and H2B genes in rainbow trout (Salmo gairdnerii)

Summary The complete nucleotide sequence of the 3.0-kb BamH I-Sst I restriction fragment contained within the rainbow trout genomic clone TH2 has been determined. This region contains the rainbow trout H4 and H2B histone genes and 5 and 3 flanking and spacer sequences, and represents the 5 half of the histone-gene cluster; the remaining half has been characterized previously. The genes are uninterrupted, and are transcribed from the same strand. The protein sequence of H4, as determined from the nucleic acid sequence, is the same as that derived for other vertebrate H4 proteins, although comparison of nucleotide sequences shows a great deal of sequence divergence, especially in the third base position. The amino acid sequence of H2B, though largely homologous to those of other vertebrate H2B proteins, displays some characteristic differences in primary structure. Consensus sequences noted in many other eukaryotic genes, as well as histone-specific consensus sequences, have been identified. An unusual feature of the spacer region between the H4 and H2B genes is the presence of a duplicated sequence 87 bp in length. The 5 and 3 ends of each repeat are complementary, and each repeat contains smaller repeated sequences internally, as well as a possible cruciform structure.