Balbiani Ring Genes Research Articles

Abrupt evolutionary change in the Balbiani ring gene family in two sibling species ofChironomus

We have investigated interspecific divergence in a eucaryotic gene family, the Balbiani ring genes of the dipteran genusChironomus. In general, our data show thatC. pallidivittatus andC. tentans possess virtually identical Balbiani ring gene sequences, whereasC. thummi represents a more distantly related member of the genus. These results are consistent with phylogenetic relationships for the three species based on polytene chromosome banding patterns. However, one Balbiani ring gene sequence displays an evolutionarily anomalous behavior. A 16-kilobase-pair Balbiani ring gene 1 sequence inC. pallidivittatus that we show to be transcribed is absent in the sibling speciesC. tentans. We propose that this sequence was deleted inC. tentans after divergence of the species. Considering the importance of Balbiani ring genes for feeding and protection of theChironomus larvae, we suggest that the pronounced interspecific genetic difference contributes to a difference in ecological niche between the two sympatrically distributed species.

Different evolutionary behavior of structurally related, repetitive sequences occurring in the same Balbiani ring gene in Chironomus tentans

The Balbiani ring 2 (BR 2) gene in Chironomus tentans is highly internally repeated. Two types of related repeat units-the alpha and beta types-are tandemly arranged in separate blocks, which together are likely to form the major part of the gene. Every repeat unit has one constant region and one subrepeat region. Here we analyze the length and sequence of a number of repeat units of both types and compare the units within and between the blocks. The approximately 100 alpha repeat units are essentially invariant regarding length and sequence. In contrast, when the approximately 70 beta repeat units are compared, six length variants are found, four of which have been sequenced. The length variations reside in the subrepeat regions and are due to different numbers of whole or half subrepeats. Furthermore, the subrepeat regions differ by several base-pair substitutions, many of which change the amino acid sequence. On the other hand, all beta-type constant regions are of equal length and are virtually homogeneous in sequence. The observed length distributions in combination with analysis of the basepair substitutions in the alpha-and beta-type constant and subrepeat regions suggest that the alpha and beta blocks are of different age, that seemingly homologous repeated regions may play different functional roles at the protein level, and that sequence correction mechanisms are likely to operate to different extents on the constant and subrepeat regions within the beta block.

Packing of a specific gene into higher order structures following repression of RNA synthesis.

Transcription of the Balbiani ring (BR) genes of the dipteran Chironomus tentans was inhibited by teh nucleoside analogue DRB (5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole). The BR genes were emptied of RNA polymerases and the subsequent packing of the genes was monitored by transmission electron microscopy. The thin chromatin axis of the transcriptionally active genes condensed into a thick (20-25 nm) chromatin fiber, which was recorded as a linear structure, an open loop or a supercoiled loop. The compacted genes were finally packed into dense clumps of chromatin. It was proposed that upon repression of RNA synthesis the BR gene template attains the following consecutive stages with increasing compaction: transcription loop----linear thick fiber----open thick fiber loop----supercoiled thick fiber loop----dense chromatin. Within the chromatin blocks structures that resembled the supercoiled loops were discerned, suggesting that the final packing of the template might be accomplished by a close alignment of supercoiled loops.

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.

Visualization of the formation and transport of a specific hnRNP particle

The growth and maturation of the transcription products on the Balbiani ring (BR) genes in Chironomus tentans has been characterized by electron microscopy. The BR transcript is packed into a series of well defined ribonucleoprotein structures of increasing complexity: a 10 nm fiber, a 19 nm fiber, a 26 nm fiber, and a 50 nm granule. The basic 10 nm element was revealed in Miller spreads. The in situ structure of the transcription products and RNA compaction estimates suggested that the 10 nm fiber is packed into the 19 nm fiber as a tight coil. The transition of the 19 nm fiber into the 26 nm fiber is accompanied by a major change of the basic 10 nm fold into a noncoiled structure. Finally, the 26 nm fiber makes a one and one-third left-handed turn forming the final product, the BR granule. Upon translocation through the nuclear pore the BR granule becomes rod-shaped, which most likely corresponds to a relaxation of the highest-order structure into a straight 26 nm fiber.

Secretory proteins and Balbiani ring gene activities in salivary glands of Chironomus thummi larvae

The salivary gland cells of Chironomus larvae synthesize almost exclusively secretory proteins, for constructing the tubes in which they live. The secretory proteins of C. thummi contain a high percentage of basic amino acids, chiefly lysine (about 20%), and consist of two main giant secretory proteins of about 1,000 kd, called sp-Ia and sp-Ib, and a number of low-molecular-weight fractions, sp-180, sp-150, sp-36, sp-18, and sp-15. Galactose treatment shows that the synthesis of sp-I fractions is correlated with the puffing of Balbiani rings, BRb and BRc, and with the relative abundance of Balbiani ring RNA transcripts in the cytoplasm. The sp-I fractions, which appear to represent real polypeptides, display individual- and strain-specific size variations. Cleavage of sp-I proteins with cyanogen bromide (CNBr) yields peptides of about 17 kd and peptide ladders composed of 8–10 multimers of this 17kd “basic sequence”. These properties are typical for proteins encoded by the giant, internally repeated Balbiani ring genes.

Rapid reformation of the thick chromosome fiber upon completion of RNA synthesis at the Balbiani ring genes in Chironomus tentans.

We have studied the ultrastructure of the Balbiani ring genes in Chironomus tentans during treatment with the RNA synthesis inhibitor DRB (5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole). This nucleoside analogue blocks transcription at or near the initiation site but does not interfere with the elongation and termination processes. In the ordinary active state the Balbiani ring genes display a 5 nm chromosome fiber, carrying densely distributed, growing ribonucleoprotein particles (Andersson et al., 1980). When the transcriptional activity declines, a 10 nm fiber can be observed between sparsely distributed RNA polymerases. Furthermore, after passage of the last RNA polymerase the 10 nm fiber can be seen as well as its gradual packing into a 25 nm thick fiber. Thus, the active chromosome fiber is rapidly packed into higher order structures when the fiber is not directly involved in transcription. The formation of the thick fiber does not require that the gene along its entire length is devoid of active RNA polymerases. The thick fiber can again be mobilized for transcription, since in reversion experiments the BR genes appear as ordinary active genes with an extended nucleofilament and densely packed nascent transcription products. The dynamic behaviour of the chromosome fiber during transcription is discussed as well as the packing and unpacking of a gene into higher order structures.

Evidence for a common ancestor sequence for the Balbiani ring 1 and Balbiani ring 2 genes in Chironomus tentans

The Balbiani ring (BR) 1 and BR 2 genes in Chironomus tentans are functionally related and are only expressed in the salivary gland cells. Here we reveal the principal structure of the BR 1 gene and analyze the structural and evolutionary relationship between the BR 1 and BR 2 genes. The properties of the BR 1 gene, 37 kilobases in size, are derived from the analysis of a cloned cDNA sequence, pCt 21. A considerable part of the BR 1 gene consists of one or a few blocks of a tandemly repeated 246-base-pair (bp) major repeat unit. About half of this major repeat unit is in turn built from four tandem repeats of a 33-bp sequence. This hierarchic arrangement of repetitive sequences within the BR 1 gene suggests that the gene has evolved through two major amplification steps, starting from a short primordial sequence. A similar evolutionary model has been put forward for the BR 2 gene [Sümegi, J., Wieslander, L. & Daneholt, B. (1982) Cell 30, 579-587]. The two putative primordial genes contain a similar, 102-bp-long sequence (86% nucleotide sequence homology), indicating that the BR 1 and BR 2 genes most likely arose from the same ancestor sequence. During the course of evolution the two genes diverged, mainly due to differences in the length and sequence of the gene segments involved in the two amplification steps. Moreover, at least one of the BR genes was translocated to another chromosomal locus.

Characterization of a 249-bp tandemly repetitive, satellite-like repeat in the translated portion of Balbiani ring c of Chironomus thummi.

A major part of Balbiani ring (BR)c DNA of Chironomus thummi consists of tandem 249-bp repeats which appear to be transcribed and translated into a polypeptide of very unusual composition. Whereas these 2549-bp repeats are evident by Southern blotting, sequence analysis reveals a finer tandemly repetitive substructure: more than half of the 249-bp repeat length consists of tandem 24-bp subrepeats , and these in turn may have been generated from even shorter sequences. Comparisons with partial BRb and BR1 sequences reveal that this hierarchically repetitive sequence structure is typical of BR genes. It resembles the structure of some satellite sequences, suggesting that mechanisms leading to satellite DNA evolution may also operate in the evolution of structural genes.

Cell-specific synthesis and glycosylation of secretory proteins in larval salivary glands of Chironomus thummi.

Synthesis and glycosylation of larval salivary gland secretory proteins of Chironomus thummi were analyzed with respect to cell specific differences in the Balbiani ring (BR) pattern and glycoprotein composition of secretion formerly detected by histochemical staining procedures. In the secretion of a special cell type in salivary glands, which is characterized by the appearance of an additional BR, and additional polypeptide with a relative molecular weight (Mr) of 160 kD was found differing in its antigenic properties and tryptic fingerprint pattern from main cell secretion proteins. This so-called ssp-160 component is preferentially synthesized and glycosylated in the special cells. In the same cells, both the synthesis and glycosylation of all other major secretory proteins was found to be diminished or even repressed. In contrast to the conspicuous cell-specific differences at the level of protein synthesis. RNA analyses show the prominent synthesis of 75S RNA in both main and special cells and gave no clear indication of the synthesis of a smaller RNA fraction as expected from the size of ssp-160 component.--These and further data on synthesis and properties of secretory proteins as well as expression of BR DNA are discussed with regard to the assumption that at least some of the eight major secretory polypeptides are coded for by BR DNA. The BR gene(s) might have originated by manifold duplications and modifications of short repetitive prototype DNA sequences, which are coordinatively expressed.

Study of salivary gland specific transcripts in Chironomus tentans by cDNA analysis.

Preparative amounts of polyA-containing RNA were purified from isolated salivary glands from Chironomus tentans and used as a template for the synthesis of cDNA by AMV reverse transcriptase. The yield of cDNA corresponded to 8-14% of the input polyA RNA. Using the Alwine hybridization method (Alwine et al., 1977) it was shown that only a minor part of the cDNA hybridized to the 75S RNA band and that the major hybridization occurred to a hitherto unknown gland-specific polyA RNA transcript of 1.3 kb length. In addition, the cDNA hybridized to a gland-specific polyA-RNA transcript of 2 kb length. - The majority of the cDNA had a size of 0.4 or 0.7 kb in equal amounts. A Southern hybridization of the cDNA to an EcoRI digest of genomic DNA showed in addition to a very weak hybridization to the 40 kb band of the Balbiani ring genes, hybridization to several restriction fragments with sizes from 1.3 to 16 kb.

Activation of Balbiani ring genes in Chironomus tentans after a pilocarpine-induced depletion of the secretory products from the salivary gland lumen

The synthesis of protein and RNA has been studied in Chironomus salivary glands after pilocarpine-induced depletion of secretory products from the gland lumen. Emptied glands showed a more than fourfold enhancement of protein synthesis, the secretory proteins being the main product. The large chromosomal puffs, Balbiani ring 1 (BR 1) and Balbiani ring 2 (BR 2), likely to be responsible for the production of messenger RNA for the secretory proteins, expanded to a considerable extent and the synthesis of the corresponding RNA species, 75 S RNA, increased fivefold after a 5-h pilocarpine treatment. In vitro experiments suggest that pilocarpine does not exert its effect on the expression of the BR genes via a direct action on the salivary gland cells themselves. The effect is more likely to result from stimulation of the retractor muscles (near the secretory duct), regulating the discharge of the secretory products from the gland lumen and, consequently, also the amount of secretory products present in the gland lumen at any given moment.

Periodicities and tandem repeats in a Balbiani ring gene

The Balbiani ring (BR) DNAs show prominent periodicities of restriction enzyme sites. Studies using a cloned fragment of the BRc gene strongly suggest that these periodicities reflect the existence of tandemly repetitive sequences within BR DNA. Tandem repeats measuring 54–58 bp have been demonstrated by partial sequence analysis of the BRc clone; the restriction site periodicities suggest the existence of additional 175 (= 3 × 58) and 1050 (= 6 × 175) bp repeat units. The short, medium and long repeats (58, 175 and 1050 bp, respectively) show sequence homology. Constrained unequal crossing over (resulting from misalignment of repeat arrays, usually by one repeat) is proposed as the mechanism for evolution of short, medium and long repeats from each other, in a manner analogous to evolution of satellite DNA sequences. Paradoxically, the dominant restriction site periodicities appear to be more conservative than might be expected on the basis of the overall sequence divergence between the sequenced repeats. This may be a consequence of functionally important, long-range amino acid or oligopeptide periodicities (for example, Asp · Ser or Glu · Ser corresponding to Hinf I sites) in the BRc protein product, in conjunction with preferential use of certain synonymous codons.