Complex Genomic Loci Research Articles

O-270 Clinical-grade whole genome sequencing-based haplarithmisis enables all forms of preimplantation genetic testing

Abstract Study question Can a simple, and scalable assay be developed to concurrently perform all forms of preimplantation genetic testing (PGT), effectively addressing the increasing demand? Summary answer Whole genome sequencing-based haplarithmisis offers a single-assay solution for all forms of PGT, enhancing resolution and diagnosis in complex cases. What is known already State-of-the-art preimplantation genetic testing (PGT) methods, including genotyping-by-sequencing (GBS)-based haplotyping, enable PGT for monogenic disorders (PGT-M), structural rearrangements (PGT-SR), and aneuploidies (PGT-A) in a single assay. However, they are labour intensive, only partially cover the genome, require a close relative to determine meiotic and mitotic origin of aneuploidies, and are troublesome for consanguineous families and in complex genomic contexts. Furthermore, the prevailing PGT approach for mitochondrial DNA disorders (PGT-MT) involves a PCR-based method. This underscores the need to develop a universal PGT method that streamlines laboratory protocols and provides genome-wide coverage to address genetic disorders, even in complex genomic loci. Study design, size, duration In a retrospective pilot study, we compared whole genome sequencing (WGS) at 30-40X with GBS (6 embryos, 2 families, 3 indications). We carried out subsampling of WGS data and determined 10X as the optimal diagnostic depth-of-coverage. We clinically validated our approach by comparing WGS to GBS for PGT-M and PGT-SR (31 embryos, 16 families, 22 indications), to shallow sequencing for PGT-SR (10 embryos, 3 families) and to PCR-based PGT for PGT-MT (10 embryos, 2 families). Participants/materials, setting, methods DNA from parents and close relative(s) and whole genome amplified (WGAed) DNA from embryos, underwent WGS with subsequent haplarithmisis. Quality metrics, such as Mendelian inconsistency rates, number of informative SNPs, and haplotype concordance were assessed. Translocation breakpoints were identified utilizing aberrantly aligned paired sequencing reads. Additionally, single-nucleotide variants (SNVs), including mitochondrial DNA SNVs, were detected by allele counting. Finally, segregational origin was determined via a parents-only haplarithmisis approach that uses the embryo as phasing reference. Main results and the role of chance WGS-PGT alleviates technical limitations by decreasing WGA artifacts by 68.4%, increasing breadth of coverage by 4-fold, and decreasing wet-lab turnaround time by 2.5-fold. Owing to its inherent higher resolution and improved genomic coverage, 5 out of 13 – previously inconclusive – embryos with genetic disorders in complex genomic regions could be assigned a diagnosis. Furthermore, we show the ability to directly detect single- and few-base pair genetic variants in any region of interest. Importantly, we show trio-based PGT-A for aneuploidy origin, an approach we term PGT-AO that – using the parents-only genotype calls – can detect the segregational origin of aneuploidies without the need of a close relative as reference for phasing. The segregational origin detected by parents-only haplarithmisis was concordant with the segregational origin determined from haplarithmisis when using a close relative. Unbalanced translocations could be detected indirectly via haplotyping or directly at single base resolution using a structural variant caller. Mitochondrial heteroplasmy level calculation from WGS yielded comparable results to PCR-based methods, both in blastomere and trophectoderm biopsy samples. Limitations, reasons for caution Direct variant detection with an average coverage of 10X may occasionally be inadequate, as certain locations may lack sufficient coverage (<5 reads). Inherently to the limited sample size of PGT-MT families, WGS-PGT needs further validation. The need for invasive trophectoderm biopsies of preimplantation embryos remains the key limitation of WGS-PGT. Wider implications of the findings Our approach enables all forms of PGT in a single assay. Direct variant detection complements haplarithmisis, addressing inconclusive findings and aiding families with a de novo variant or lacking a close relative for phasing. Finally, PGT-AO facilitates research on the origin of aneuploidies in preimplantation embryos and their clinical outcome. Trial registration number not applicable

Molecular mechanisms protecting centromeres from self-sabotage and implications for cancer therapy.

Centromeres play a crucial role in DNA segregation by mediating the cohesion and separation of sister chromatids during cell division. Centromere dysfunction, breakage or compromised centromeric integrity can generate aneuploidies and chromosomal instability, which are cellular features associated with cancer initiation and progression. Maintaining centromere integrity is thus essential for genome stability. However, the centromere itself is prone to DNA breaks, likely due to its intrinsically fragile nature. Centromeres are complex genomic loci that are composed of highly repetitive DNA sequences and secondary structures and require the recruitment and homeostasis of a centromere-associated protein network. The molecular mechanisms engaged to preserve centromere inherent structure and respond to centromeric damage are not fully understood and remain a subject of ongoing research. In this article, we provide a review of the currently known factors that contribute to centromeric dysfunction and the molecular mechanisms that mitigate the impact of centromere damage on genome stability. Finally, we discuss the potential therapeutic strategies that could arise from a deeper understanding of the mechanisms preserving centromere integrity.

Long Non-coding RNAs and CRISPR-Cas Edition in Tumorigenesis.

Long non-coding RNAs (lncRNAs) are one of the most abundant and heterogeneous transcripts with key roles in chromatin remodeling and gene regulation at the transcriptional and post-transcriptional levels. Due to their role in cell growth and differentiation, lncRNAs have emerged as an important biomarker in cancer diagnosis, prognosis, and targeted treatment. Recent studies have focused on elucidating lncRNA function during malignant transformation, tumor progression and drug resistance. The advent of the CRISPR system has made it possible to precisely edit complex genomic loci such as lncRNAs. Thus, we summarized the advances in CRISPR-Cas approaches for functional studies of lncRNAs including gene knockout, knockdown, overexpression and RNA targeting in tumorigenesis and drug resistance. Additionally, we highlighted the perspectives and potential applications of CRISPR approaches to treat cancer, as an emerging and promising target therapy.

Pathways to polar adaptation in fishes revealed by long-read sequencing.

Long-read sequencing is driving a new reality for genome science in which highly contiguous assemblies can be produced efficiently with modest resources. Genome assemblies from long-read sequences are particularly exciting for understanding the evolution of complex genomic regions that are often difficult to assemble. In this study, we utilized long-read sequencing data to generate a high-quality genome assembly for an Antarctic eelpout, Ophthalmolycus amberensis, the first for the globally distributed family Zoarcidae. We used this assembly to understand how O. amberensis has adapted to the harsh Southern Ocean and compared it to another group of Antarctic fishes: the notothenioids. We showed that selection has largely acted on different targets in eelpouts relative to notothenioids. However, we did find some overlap; in both groups, genes involved in membrane structure, thermal tolerance and vision have evidence of positive selection. We found evidence for historical shifts of transposable element activity in O. amberensis and other polar fishes, perhaps reflecting a response to environmental change. We were specifically interested in the evolution of two complex genomic loci known to underlie key adaptations to polar seas: haemoglobin and antifreeze proteins (AFPs). We observed unique evolution of the haemoglobin MN cluster in eelpouts and related fishes in the suborder Zoarcoidei relative to other Perciformes. For AFPs, we identified the first species in the suborder with no evidence of afpIII sequences (Cebidichthys violaceus) in the genomic region where they are found in all other Zoarcoidei, potentially reflecting a lineage-specific loss of this cluster. Beyond polar fishes, our results highlight the power of long-read sequencing to understand genome evolution.

Whole blood transcriptomic profiles can differentiate vulnerability to chronic low back pain.

The mechanisms underlying the transition from acute to chronic pain remain unclear. Here, we sought to characterize the transcriptome associated with chronic low back pain as well as the transcriptome of the transition from acute to chronic low back pain. For the analysis, we compared the whole blood transcriptome of: (a) patients at the onset of low back pain who no longer had pain within 6 weeks after onset (acute) with patients who developed chronic low back pain at 6 months (chronic T5); and, (b) patients at the onset of low back pain (chronic T1) who developed chronic pain at 6 months with healthy pain-free (normal) controls. The majority of differentially expressed genes were protein coding. We illustrate a unique chronic low back pain transcriptome characterized by significant enrichment for known pain genes, extracellular matrix genes, and genes from the extended major histocompatibility complex (MHC) genomic locus. The transcriptome of the transition from acute to chronic low back pain was characterized by significant upregulation of antigen presentation pathway (MHC class I and II) genes and downregulation of mitochondrial genes associated with oxidative phosphorylation, suggesting a unique genomic signature of vulnerability to low back pain chronicity.

Revisiting Dscam diversity: lessons from clustered protocadherins.

The complexity of neuronal wiring relies on the extraordinary recognition diversity of cell surface molecules. Drosophila Dscam1 and vertebrate clustered protocadherins (Pcdhs) are two classic examples of the striking diversity from a complex genomic locus, wherein the former encodes more than 10,000 distinct isoforms via alternative splicing, while the latter employs alternative promoters to attain isoform diversity. These structurally unrelated families show remarkably striking molecular parallels and even similar functions. Recent studies revealed a novel Dscam gene family with tandemly arrayed 5' cassettes in Chelicerata (e.g., the scorpion Mesobuthus martensii and the tick Ixodes scapularis), similar to vertebrate clustered Pcdhs. Likewise, octopus shows a more remarkable expansion of the Pcdh isoform repertoire than human. These discoveries of Dscam and Pcdh diversification reshape the evolutionary landscape of recognition molecule diversity and provide a greater understanding of convergent molecular strategies for isoform diversity. This article reviews new insights into the evolution, regulatory mechanisms, and functions of Dscam and Pcdh isoform diversity. In particular, the convergence of clustered Dscams and Pcdhs is highlighted.

Polymer Simulations of Heteromorphic Chromatin Predict the 3D Folding of Complex Genomic Loci

SummaryChromatin folded into 3D macromolecular structures is often analyzed by chromosome conformation capture (3C) and fluorescence in situ hybridization (FISH) techniques, but these frequently provide contradictory results. Chromatin can be modeled as a simple polymer composed of a connected chain of units. By embedding data for epigenetic marks (H3K27ac), chromatin accessibility (assay for transposase-accessible chromatin using sequencing [ATAC-seq]), and structural anchors (CCCTC-binding factor [CTCF]), we developed a highly predictive heteromorphic polymer (HiP-HoP) model, where the chromatin fiber varied along its length; combined with diffusing protein bridges and loop extrusion, this model predicted the 3D organization of genomic loci at a population and single-cell level. The model was validated at several gene loci, including the complex Pax6 gene, and was able to determine locus conformations across cell types with varying levels of transcriptional activity and explain different mechanisms of enhancer use. Minimal a priori knowledge of epigenetic marks is sufficient to recapitulate complex genomic loci in 3D and enable predictions of chromatin folding paths.

Functional characterization of the mouse Serpina1 paralog DOM-7.

The generation of authentic mouse-models for human α1-antitrypsin (A1AT)-deficiency is difficult due to the high complexity of the mouse Serpina1 gene locus. Depending on the exact mouse strain, three to five paralogs are expressed, with different proteinase inhibitory properties. Nowadays with CRISPR-technology, genome editing of complex genomic loci is feasible and could be employed for the generation of A1AT-deficiency mouse models. In preparation of a CRISPR/Cas9-based genome-engineering approach we identified cDNA clones with a functional CDS for the Serpina1-paralog DOM-7. Here, we show that DOM-7 functionally inhibits neutrophil elastase (ELANE) and chymotrypsin, and therefore needs to be considered when aiming at the generation of A1AT-deficient models.

The 100-genomes strains, an S. cerevisiae resource that illuminates its natural phenotypic and genotypic variation and emergence as an opportunistic pathogen.

Saccharomyces cerevisiae, a well-established model for species as diverse as humans and pathogenic fungi, is more recently a model for population and quantitative genetics. S. cerevisiae is found in multiple environments—one of which is the human body—as an opportunistic pathogen. To aid in the understanding of the S. cerevisiae population and quantitative genetics, as well as its emergence as an opportunistic pathogen, we sequenced, de novo assembled, and extensively manually edited and annotated the genomes of 93 S. cerevisiae strains from multiple geographic and environmental origins, including many clinical origin strains. These 93 S. cerevisiae strains, the genomes of which are near-reference quality, together with seven previously sequenced strains, constitute a novel genetic resource, the “100-genomes” strains. Our sequencing coverage, high-quality assemblies, and annotation provide unprecedented opportunities for detailed interrogation of complex genomic loci, examples of which we demonstrate. We found most phenotypic variation to be quantitative and identified population, genotype, and phenotype associations. Importantly, we identified clinical origin associations. For example, we found that an introgressed PDR5 was present exclusively in clinical origin mosaic group strains; that the mosaic group was significantly enriched for clinical origin strains; and that clinical origin strains were much more copper resistant, suggesting that copper resistance contributes to fitness in the human host. The 100-genomes strains are a novel, multipurpose resource to advance the study of S. cerevisiae population genetics, quantitative genetics, and the emergence of an opportunistic pathogen.

Splinkerette PCR for Mapping Transposable Elements in Drosophila

Transposable elements (such as the P-element and piggyBac) have been used to introduce thousands of transgenic constructs into the Drosophila genome. These transgenic constructs serve many roles, from assaying gene/cell function, to controlling chromosome arm rearrangement. Knowing the precise genomic insertion site for the transposable element is often desired. This enables identification of genomic enhancer regions trapped by an enhancer trap, identification of the gene mutated by a transposon insertion, or simplifying recombination experiments. The most commonly used transgene mapping method is inverse PCR (iPCR). Although usually effective, limitations with iPCR hinder its ability to isolate flanking genomic DNA in complex genomic loci, such as those that contain natural transposons. Here we report the adaptation of the splinkerette PCR (spPCR) method for the isolation of flanking genomic DNA of any P-element or piggyBac. We report a simple and detailed protocol for spPCR. We use spPCR to 1) map a GAL4 enhancer trap located inside a natural transposon, pinpointing a master regulatory region for olfactory neuron expression in the brain; and 2) map all commonly used centromeric FRT insertion sites. The ease, efficiency, and efficacy of spPCR could make it a favored choice for the mapping of transposable element in Drosophila.

Complex sense-antisense architecture of TNFAIP1/POLDIP2 on 17q11.2 represents a novel transcriptional structural-functional gene module involved in breast cancer progression

BackgroundA sense-antisense gene pair (SAGP) is a gene pair where two oppositely transcribed genes share a common nucleotide sequence region. In eukaryotic genomes, SAGPs can be organized in complex sense-antisense architectures (CSAGAs) in which at least one sense gene shares loci with two or more antisense partners. As shown in several case studies, SAGPs may be involved in cancers, neurological diseases and complex syndromes. However, CSAGAs have not yet been characterized in the context of human disease or cancer.ResultsWe characterize five genes (TMEM97, IFT20, TNFAIP1, POLDIP2 and TMEM199) organized in a CSAGA on 17q11.2 (we term this the TNFAIP1/POLDIP2 CSAGA) and demonstrate their strong and reproducible co-regulatory transcription pattern in breast cancer tumours. Genes of the TNFAIP1/POLDIP2 CSAGA are located inside the smallest region of recurrent amplification on 17q11.2 and their expression profile correlates with the DNA copy number of the region. Survival analysis of a group of 410 breast cancer patients revealed significant survival-associated individual genes and gene pairs in the TNFAIP1/POLDIP2 CSAGA. Moreover, several of the gene pairs associated with survival, demonstrated synergistic effects. Expression of genes-members of the TNFAIP1/POLDIP2 CSAGA also strongly correlated with expression of genes of ERBB2 core region of recurrent amplification on 17q12. We clearly demonstrate that the observed co-regulatory transcription profile of the TNFAIP1/POLDIP2 CSAGA is maintained not only by a DNA amplification mechanism, but also by chromatin remodelling and local transcription activation.ConclusionWe have identified a novel TNFAIP1/POLDIP2 CSAGA and characterized its co-regulatory transcription profile in cancerous breast tissues. We suggest that the TNFAIP1/POLDIP2 CSAGA represents a clinically significant transcriptional structural-functional gene module associated with amplification of the genomic region on 17q11.2 and correlated with expression ERBB2 amplicon core genes in breast cancer. Co-expression pattern of this module correlates with histological grades and a poor prognosis in breast cancer when over-expressed. TNFAIP1/POLDIP2 CSAGA maps the risks of breast cancer relapse onto the complex genomic locus on 17q11.2.

EVI1 Is Activated During the Generation of Induced Pluripotent Stem (iPS) Cells.

Abstract 5048EVI1 gene was first identified as a common site of retroviral integration in murine leukemia models. This gene is part of a complex genomic locus, MDS1-EVI1, that has been described as a target for retroviral integration that may lead to the emergence of a non-malignant dominant hematopoietic stem cell (HSC) clone in mice, in primates, and in humans. These studies suggested that one of the genes encoded by this locus could affect the self-renewal potential of HSC. Recent studies in mice revealed that indeed EVI1 plays an essential role in cell proliferation and it also enhances the self-renewal ability of HSC. The intense attention focused on the MDS1-EVI1 locus as retrovirus integration site prompted us to investigate whether EVI1 might have a role in somatic cell re-programming generated with retroviruses. Recent developments in stem cell research have enabled the re-programming of somatic cells to a pluripotent state using exogenous factors. Induced pluripotent stem (iPS) cells have the potential to differentiate into any cells types and that might be used in the future for clinical therapy. In order to elucidate the molecular events allowing the conversion of adult somatic into pluripotent stem cell, we evaluated EVI1 expression during this process. We found that EVI1 is activated in the early stages of re-programming and then it is silenced once the cells has been fully re-programmed. EVI1 seems to facilitate the initiation of cell re-programming by up-regulating a subset of genes previously described as potent stimulators of stem cells expansion. DisclosuresNo relevant conflicts of interest to declare.

Sequence and Characterization of the Ig Heavy Chain Constant and Partial Variable Region of the Mouse Strain 129S1

Although the entire mouse genome has been sequenced, there remain challenges concerning the elucidation of particular complex and polymorphic genomic loci. In the murine Igh locus, different haplotypes exist in different inbred mouse strains. For example, the Igh(b) haplotype sequence of the Mouse Genome Project strain C57BL/6 differs considerably from the Igh(a) haplotype of BALB/c, which has been widely used in the analyses of Ab responses. We have sequenced and annotated the 3' half of the Igh(a) locus of 129S1/SvImJ, covering the C(H) region and approximately half of the V(H) region. This sequence comprises 128 V(H) genes, of which 49 are judged to be functional. The comparison of the Igh(a) sequence with the homologous Igh(b) region from C57BL/6 revealed two major expansions in the germline repertoire of Igh(a). In addition, we found smaller haplotype-specific differences like the duplication of five V(H) genes in the Igh(a) locus. We generated a V(H) allele table by comparing the individual V(H) genes of both haplotypes. Surprisingly, the number and position of D(H) genes in the 129S1 strain differs not only from the sequence of C57BL/6 but also from the map published for BALB/c. Taken together, the contiguous genomic sequence of the 3' part of the Igh(a) locus allows a detailed view of the recent evolution of this highly dynamic locus in the mouse.

Combinatorial Expression of α- and γ-Protocadherins Alters Their Presenilin-Dependent Processing

Alpha- and gamma-protocadherins (Pcdhs) are type I transmembrane receptors expressed predominantly in the central nervous system and located in part in synapses. They are transcribed from complex genomic loci, giving rise in the mouse to 14 alpha-Pcdh and 22 gamma-Pcdh isoforms consisting of variable domains, each encompassing the extracellular region, the transmembrane region, and part of the intracellular region harboring the alpha- or gamma-Pcdh-specific invariant cytoplasmic domain. Presenilin-dependent intramembrane proteolysis (PS-IP) of gamma-Pcdhs and the formation of alpha/gamma-Pcdh heteromers led us to investigate the effects of homo- and heteromer formation on gamma- and putative alpha-Pcdh membrane processing and signaling. We find that upon surface delivery, alpha-Pcdhs, like gamma-Pcdhs, are subject to matrix metallo-protease cleavage followed by PS-IP in neurons. We further demonstrate that the combinatorial expression of alpha- and gamma-Pcdhs modulates the extent of their PS-IP, indicating the formation of alpha/gamma-Pcdh heteromers with an altered susceptibility to processing. Cell-specific expression of alpha/gamma-Pcdh isoforms could thus determine cell and synapse adhesive properties as well as intracellular and nuclear signaling by their soluble cytoplasmic cleavage products, alpha C-terminal fragment 2 (alpha-CTF-2) and gamma-CTF-2.

Chromatin Immunoprecipitation in the Analysis of Large Chromatin Domains Across Murine Antigen Receptor Loci

This chapter provides a full, detailed description of chromatin immunoprecipitation (ChIP) method, including the subsequent analysis of the immunoprecipitated DNA. ChIP is a powerful technique used to analyze protein or DNA interactions in a native chromatin context. In conjunction with previously favored procedures such as nuclease sensitivity mapping, ChIP also provides an additional tool to search for specific DNA regulatory elements. The chapter presents a ChIP protocol used to identify the histone modification patterns at murine immunoglobulin heavy chain (IgH) locus. Data generated from ChIP experiments provides valuable insight into how specific gene segments within a locus are made accessible to the recombination machinery while simultaneously restricting access to other gene segments during lymphocyte development. The ChIP method involves three basic steps, namely— (1) In vivo formaldehyde crosslinking of intact cells, (2) Micrococcal nuclease (MNase) digestion and sonication of the chromatin to generate a pool of small chromatin fragments, and (3) Selective immunoprecipitation (IP) of protein or DNA complexes from the chromatin pool with specific antibodies. The ChIP method described in this chapter provides another versatile tool, in addition to DNaseI hypersensitivity mapping, which can be used in a number of unique cell types to identify regulatory elements, such as promoters, enhancers, or boundary elements as well as define the histone modification pattern across chromatin sub-domains within a large, complex genomic locus.

Onchocerca volvulus: identification of cDNAs encoding a putative phosphatidyl-ethanolamine-binding protein and a putative partially processed mRNA precursor

We have identified and sequenced cDNAs of the parasitic nematode Onchocerca volvulus which encode a homologue of phosphatidylethanolamine-binding proteins from mammals. These clones are also closely related to the O. volvulus Ov16 cDNA (Lobos et al., 1990). One identified cDNA clone appears to represent a partially processed precursor. These results suggest that these cDNAs are derived from a complex genomic locus, raising the possibility of polycistronic transcription in this nematode.

Gene expression from the c-erbA alpha/Rev-ErbA alpha genomic locus. Potential regulation of alternative splicing by opposite strand transcription.

Alternative splicing of the c-erbA alpha gene transcript generates mRNAs encoding a thyroid hormone (T3) receptor (alpha 1) and a non-T3-binding variant (alpha 2). Also encoded at this genomic locus is Rev-ErbA alpha (Rev), a non-T3-binding member of the T3/steroid hormone receptor family. The DNA strand coding for Rev is opposite of that encoding the c-erbA alpha proteins, such that bidirectional transcription is required to generate the Rev and alpha 2 mRNAs. We have used cycloheximide as a tool for studying the regulation of gene expression from this complex genomic locus. In 235-1 cells cycloheximide treatment increased Rev mRNA levels by 50-100-fold. The effect was detectable after a 1-2-h incubation with 20 micrograms/ml cycloheximide and maximal at 24 h. The cycloheximide-induced increase in Rev gene expression was due to inhibition of protein synthesis since anisomycin caused a similar induction of Rev mRNA. Nuclear run-on assays revealed an approximately 10-fold increase in the transcriptional rate of the Rev gene during cycloheximide treatment. In addition, cycloheximide stabilized the Rev mRNA, as evidenced by half-life determinations in the presence of actinomycin D (5 ng/ml). Thus, labile proteins repress basal Rev gene expression both transcriptionally and post-transcriptionally. Cycloheximide treatment also increased alpha 1 and decreased alpha 2 mRNA levels, with the alpha 1/alpha 2 ratio increasing approximately 10-fold. Interestingly, however, these effects appeared not to be due to changes in rates of transcription or degradation of either mRNA. A likely alternative, given the structure of the c-erbA alpha gene, is that inhibition of protein synthesis resulted in a change in the c-erbA alpha splicing pattern. We hypothesize that this may be causally related to the accumulation of Rev mRNA (or the nascent Rev gene transcript), which could inhibit splicing to alpha 2 by hybridizing to complementary sequences, favoring alpha 1 mRNA production. Since alpha 1 is a T3 receptor whereas alpha 2 is an inhibitor of T3 action, such changes could influence target cell responsiveness to T3.

Tandemly repeated exons encode 81-base repeats in multiple, developmentally regulated Schistosoma mansoni transcripts.

The adult Schistosoma mansoni cDNA clone 10-3 encodes an antigen that is recognized by sera from infected humans. We characterized multiple developmentally regulated transcripts homologous to the 10-3 cDNA and portions of the complex genomic loci encoding those transcripts. Transcripts of approximately 950, 870, and 780 nucleotides were expressed in adults, whereas only the 780-nucleotide transcript was observed in the larval stage. These transcripts were highly similar, containing variable numbers of identical direct tandem repeats of 81 bases. Although the sequence of the repeating elements and sequences 3' to them were identical in all the transcripts, sequences 5' of the repeating elements exhibited variations, including a 27-base insertion, alternative start sites for transcription, and alternate 5' exon usage. These transcripts appeared to be derived in part by the developmentally controlled alternative splicing of small exons and the use of alternative transcription initiation sites from the one or two complex loci of at least 40 kilobase pairs. Each 81-base repeat in the transcripts was encoded by three dispersed 27-base-pair exons. These 27-base-pair exons were contained within highly conserved, reiterated 3-kilobase-pair genomic tandem arrays.

Tandemly repeated exons encode 81-base repeats in multiple, developmentally regulated Schistosoma mansoni transcripts.

The adult Schistosoma mansoni cDNA clone 10-3 encodes an antigen that is recognized by sera from infected humans. We characterized multiple developmentally regulated transcripts homologous to the 10-3 cDNA and portions of the complex genomic loci encoding those transcripts. Transcripts of approximately 950, 870, and 780 nucleotides were expressed in adults, whereas only the 780-nucleotide transcript was observed in the larval stage. These transcripts were highly similar, containing variable numbers of identical direct tandem repeats of 81 bases. Although the sequence of the repeating elements and sequences 3' to them were identical in all the transcripts, sequences 5' of the repeating elements exhibited variations, including a 27-base insertion, alternative start sites for transcription, and alternate 5' exon usage. These transcripts appeared to be derived in part by the developmentally controlled alternative splicing of small exons and the use of alternative transcription initiation sites from the one or two complex loci of at least 40 kilobase pairs. Each 81-base repeat in the transcripts was encoded by three dispersed 27-base-pair exons. These 27-base-pair exons were contained within highly conserved, reiterated 3-kilobase-pair genomic tandem arrays.