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L Wieslander

Publications and source records attributed to L Wieslander.

At least 19 recordsLinked to original sources
PubMed2005

Expressed sequence tags from the midgut and an epithelial cell line of Chironomus tentans: annotation, bioinformatic classification of unknown transcripts and analysis of expression levels.

Expressed sequence tags (ESTs) were generated from two Chironomus tentans cDNA libraries, constructed from an embryo epithelial cell line and from larva midgut tissue. 8584 5'-end ESTs were generated and assembled into 3110 tentative unique transcripts, providing the largest contribution of C. tentans sequences to public databases to date. Annotation using Blast gave 1975 (63.5%) transcripts with a significant match in the major gene/protein databases, 1170 with a best match to Anopheles gambiae and 480 to Drosophila melanogaster. 1091 transcripts (35.1%) had no match to any database. Studies of open reading frames suggest that at least 323 of these contain a coding sequence, indicating that a large proportion of the genes in C. tentans belong to previously unknown gene families.

Animals↗
PubMed2001

In situ transcription and splicing in the Balbiani ring 3 gene.

The Balbiani ring 3 (BR3) gene contains 38 introns, and more than half of them are co-transcriptionally excised. We have determined the in situ structure of the active BR3 gene by electron tomography. Each of the 20-25 nascent transcripts on the gene is present together with splicing factors and the RNA polymerase II in a nascent transcript and splicing complex, here called the NTS complex. The results indicate that extensive changes in overall shape, substructure and molecular mass take place repeatedly within an NTS complex as it moves along the gene. The volume and calculated mass of the NTS complexes show that, maximally, one complete spliceosome is assembled on the multi-intron transcript at any given time point. The structural data show that the spliceosome is not a structurally well-defined unit in situ and that the C-terminal domain of the elongating RNA polymerase II cannot carry spliceosomal components for all introns in the BR3 transcript. Our data indicate that spliceosomal factors are continuously added to and released from the NTS complexes during transcription elongation.

Animals↗
PubMed1999

The intranuclear movement of Balbiani ring premessenger ribonucleoprotein particles.

Specific premessenger ribonucleoprotein (pre-mRNP) particles, the Balbiani ring (BR) granules in the salivary glands of the dipteran Chironomus tentans, can be visualized in the electron microscope when they assemble on the genes, move through nucleoplasm, and bind to and translocate through the nuclear pores. As shown by BrUTP labeling and immunoelectron microscopy, newly synthesized BR RNP particles, released from the BR genes, appear early in all nucleoplasmic regions of the cell nucleus and they saturate the nucleoplasmic pool of BR particles after 2 h of labelling. It is concluded that within the nucleus the BR particles move randomly. Furthermore, estimates of minimum diffusion coefficients for the BR particles are compatible with the view that the particles diffuse freely in the interchromosomal space, although it is not excluded that the random movement could be slightly retarded. Once the particles get bound to the nuclear pore complexes, they seem committed to translocation through the nuclear pores.

Animals↗
PubMed1998

Transcriptional termination in the Balbiani ring 1 gene is closely coupled to 3'-end formation and excision of the 3'-terminal intron.

We have analyzed transcription termination, 3'-end formation, and excision of the 3'-terminal intron in vivo in the Balbiani ring 1 (BR1) gene and its pre-mRNA. We show that full-length RNA transcripts are evenly spaced on the gene from a position 300 bp upstream to a region 500-700 bp downstream of the polyadenylation sequence. Very few full-length transcripts and no short, cleaved, nascent transcripts could be observed downstream of this region. Pre-mRNA with 10-20 adenylate residues accumulates at the active gene and then rapidly leaves from the gene locus. Only polyadenylated pre-mRNAs could be detected in the nucleoplasm. Our results are consistent with the hypothesis that transcription termination occurs in a narrow region for the majority of transcripts, simultaneous with 3'-end formation. Excision of the 3'-terminal intron occurs before 3'-end formation in about 5% of the nascent transcripts. When transcription terminates, 3' cleavage takes place and 10-20 adenylate residues are added, the 3'-terminal intron is excised from additionally about 75% of the pre-mRNA at the gene locus. Our data support a close temporal and spatial coupling of transcription termination and the cleavage and initial polyadenylation of 3'-end formation. Excision of the 3'-terminal intron is highly stimulated as the cleavage/polyadenylation complex assembles and 3'-end formation is initiated.

Animals↗
PubMed1998

Promoter regions of four Balbiani ring genes in Chironomus tentans exhibit a common salivary gland-specific chromatin organisation, which is independent of the rate of transcriptional initiation.

The Balbiani ring (BR) gene family consists of four closely related genes which show some differences in transcriptional regulation. Here we analyse the chromatin structure of the promoter regions of the four BR genes. In cells in which the genes are inactive, digestion of chromatin with micrococcal nuclease suggests for the BR1 gene that precisely positioned nucleosomes are present from position +200 to -600. In salivary gland cells, a distinctly different organisation of the promoter chromatin leads to the appearance of two sites that are hypersensitive to both DNase I and micrococcal nuclease, located at the approximate positions -50 and -240. Nuclease digestion experiments suggest the presence of a nucleosome in the region between the two hypersensitive sites, and nucleosome disruption at the transcription initiation site. The positions of these hypersensitive sites in all BR genes is unaffected by extensive modulation of the rate of transcriptional initiation, although some alterations in the intensity of the hypersensitive sites were observed. We also show that total nucleosome displacement from the transcribing template is seen only when the RNA polymerase density exceeds one polymerase molecule per 100 bp.

Animals↗
PubMed1997

Using PRINS for gene mapping in polytene chromosomes.

We have adapted the primed in situ labelling (PRINS) protocol for gene mapping in polytene chromosomes of two dipteran species. The method was used to localize the genes for the Balbiani ring (BR) 2.1 and the iron-regulatory protein 1A (IRP1A) in polytene salivary gland chromosomes of Chironomus tentans, and Drosophila melanogaster respectively. Two oligonucleotides, corresponding to the BR 2.1 and IRP1A genes, were used as primers and the whole procedure was performed within 3-4 h. The strong labelling with low background revealed the localization of the BR 2.1 gene in polytene chromosome IV of C. tentans and the IRP1A gene in polytene chromosome 3R83 of D. melanogaster. The results demonstrated that PRINS is a fast, sensitive and suitable approach for physical gene mapping in polytene chromosomes.

Animals↗
PubMed1996

Processing of pre-mRNA in polytene nuclei of Chironomus tentans salivary gland cells.

Salivary gland polytene cells in the dipteran Chironomus tentans provide exceptional experimental possibilities to analyze processing of specific pre-mRNAs in intact eukaryotic cell nuclei. Here we give a brief account of how these experimental advantages can be exploited to analyze the splicing process in vivo. In multi-intron pre-mRNAs, spliceosomes assemble and splicing is initiated cotranscriptionally for all introns. Intron excision may, however, occur mainly co- transcriptionally or mainly posttranscriptionally depending on the position of each intron in relation to the remaining transcription time and intron-specific efficiencies of excision. As measured for the U2 snRNP and an SR protein, 10-15% of the spliceosomal components are bound to pre-mRNA at active gene loci at a given moment, while the majority of the spliceosomal components are present in the nucleoplasm. A continuous redistribution of the spliceosomal components takes place in the nucleus as a result of a close coupling between transcription and spliceosomal assembly.

Animals↗
PubMed1996

Demonstration of a dynamic, transcription-dependent organization of pre-mRNA splicing factors in polytene nuclei.

We describe the dynamic organization of pre-mRNA splicing factors in the intact polytene nuclei of the dipteran Chironomus tentans. The snRNPs and an SR non-snRNP splicing factor are present in excess, mainly distributed throughout the interchromatin. Approximately 10% of the U2 snRNP and an SR non-snRNP splicing factor are associated with the chromosomes, highly enriched in active gene loci where they are bound to RNA. We demonstrate that the splicing factors are specifically recruited to a defined gene upon induction of transcription during physiological conditions. Concomitantly, the splicing factors leave gene loci in which transcription is turned off. We also demonstrated that upon general transcription inhibition, the splicing factors redistribute from active gene loci to the interchromatin. Our findings demonstrate the dynamic intranuclear organization of splicing factors and a tight linkage between transcription and the intranuclear organization of the splicing machinery.

Animals↗
PubMed1996

The intranuclear site of excision of each intron in Balbiani ring 3 pre-mRNA is influenced by the time remaining to transcription termination and different excision efficiencies for the various introns.

The 10.9-kb Balbiani ring 3 (BR3) gene contains 38 constitutively excised introns. Both nascent and nucleoplasmic, released BR3 gene pre-mRNA can be isolated by microdissection of the polytene salivary gland nuclei in which the gene is transcribed. Here we analyze the order of intron excision in relation to transcription and to intranuclear transport. We demonstrate that the introns are excised with an overall 5' to 3' polarity that is established during transcription and maintained during transport. In contrast, we also show that individual introns are excised at very different rates and that neighboring introns are removed in a preferred order that is not necessarily 5' to 3'. Splicing factors are, in addition, shown to associate with the nascent BR3 pre-mRNA. Our data argue that functional spliceosomes assemble rapidly as introns appear in the pre-mRNA, but that intron-specific properties influence the kinetics of spliceosome assembly and/or function, resulting in cotranscriptional excision of some introns, preferentially those located in the 5' part of the pre-mRNA, and posttranscriptional excision of other introns, preferentially those located in the 3' part of the pre-mRNA.

Animals↗
PubMed1994

Splicing of Balbiani ring 1 gene pre-mRNA occurs simultaneously with transcription.

The 40 kb Balbiani ring 1 (BR1) gene is at a given moment transcribed by, on average, 120 RNA polymerases. Here we directly assay the excision of introns both in the nascent and in the released nucleoplasmic BR1 pre-mRNAs, isolated by microdissection. We show that intron 3, located 3 kb from the 5' end of the pre-mRNA, is excised simultaneous with transcription. Within 2.5 min of transcription time, 50% of the pre-mRNA molecules have lost the intron. Intron 4, located 600 bases from the polyadenylation site, is excised cotranscriptionally in 5%-10% of the molecules and after or during release to the nucleoplasm in the remaining molecules. Our results demonstrate that spliceosome assembly is a cotranscriptional process in vivo and that splicing may occur during transcription but also after completed transcription, depending on the position of the intron.

Animals↗
PubMed1994

Structure of the smallest salivary-gland secretory protein gene in Chironomus tentans.

The salivary gland secretion in the dipteran Chironomus tentans is composed of approximately 15 different secretory proteins. The most well known of the corresponding genes are the four closely related Balbiani ring (BR) genes, in which the main part of each approximately 40-kb gene is composed of tandemly arranged repetitive units. Six of the seven additional secretory protein genes described share structural similarities with the BR genes and are members of the same BR multigene family. Here we report the identification of a new secretory protein gene, the sp12 gene, encoding the smallest component of the C. tentans salivary gland secretion. The gene has a corresponding mRNA length of approximately 0.7 kb and codes for a protein with a calculated molecular weight of 7,619 Da. The sp12 gene was characterized in seven Chironomus species. Based on a comparison of the orthologous gene sequences, we conclude that the sp12 gene has a repetitive structure consisting of diverged 21-bp-long repeats. The repeat structure and the codon composition are similar to the so-called SR regions of the BR genes and the sp12 gene may represent a diverged member of the BR multigene family.

Amino Acid Sequence↗
PubMed1993

A repetitive secretory protein gene of a novel type in Chironomus tentans is specifically expressed in the salivary glands and exhibits extensive length polymorphism.

The secretion from the salivary glands in the dipteran Chironomus tentans has previously been shown to contain secretory proteins encoded by a set of related genes belonging to the Balbiani ring multigene family. Here we describe the characterization of a gene for an additional secretory protein of a novel type. This sp240/420 gene is built from virtually identical 477-base pair repeats organized in tandem. The gene exhibits extensive length polymorphism since allelic length variants with from 12 to 22 repeats were recorded. Antibodies have been raised against the gene product, and it is shown also that the protein varies in size between 240 and 420 kDa. The protein is rich in serine and threonine residues (30%) and has N-linked carbohydrate chains, presumably on each repeat. Despite careful preparation of the gland lumen proteins, the sp240/420 protein was found to form protein ladders of regularly degraded protein, suggesting that this degradation occurs naturally in the salivary glands. Although all previously characterized salivary gland secretory protein genes in C. tentans belong to the same multigene family, the sp240/420 gene appears to represent a novel, unrelated type of repetitive gene. The chromosomal location of the gene was mapped and was found to reside in region 17 on chromosome I.

Amino Acid Sequence↗
PubMed1993

Two secretory protein genes in Chironomus tentans have arisen by gene duplication and exhibit different developmental expression patterns.

The salivary gland cells in the dipteran Chironomus tentans produce approximately 15 different secretory proteins, with relative molecular masses ranging between 1 x 10(4) and 1 x 10(6). Together these proteins form two types of extra corporal tubes, a larval protective housing and feeding tube or a pupation tube. The developmental change in tube formation is accompanied by a switch in production from one combination of secretory proteins to another. Here we characterize two genes, the sp38-40.A and B genes, which encode secretory proteins with relative molecular masses of 38,000 to 40,000. The two genes are located 346 base-pairs apart in the same orientation and have presumably arisen by gene duplication as the result of an illegitimate recombination event. Both genes contain two regions with cysteine codons, surrounded by regions with short repeats coding for proline and charged amino acid residues. The two genes and alleles of the genes differ in their number of repeats. This structure resembles the structure of the Balbiani ring (BR) genes, which encode the four largest salivary gland secretory proteins. The sp38-40.A and B genes are therefore likely to belong to a BR multigene family containing all or most of the 15 salivary gland secretory protein genes. The expression of the sp38-40.A and B genes are different: the A gene is expressed throughout the larval fourth instar but considerably less in the prepupal stage, while the B gene shows the opposite expression pattern. The developmental regulation of the expression of the two genes has therefore diverged after the gene duplication event.

Amino Acid Sequence↗
PubMed1993

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 sp17 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.

Amino Acid Sequence↗