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Genes (Basel)
2024 Feb 09;152:. doi: 10.3390/genes15020222.
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Local Genomic Instability of the SpTransformer Gene Family in the Purple Sea Urchin Inferred from BAC Insert Deletions.
Barela Hudgell MA
,
Momtaz F
,
Jafri A
,
Alekseyev MA
,
Smith LC
.
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The SpTransformer (SpTrf) gene family in the purple sea urchin, Strongylocentrotus purpuratus, encodes immune response proteins. The genes are clustered, surrounded by short tandem repeats, and some are present in genomic segmental duplications. The genes share regions of sequence and include repeats in the coding exon. This complex structure is consistent with putative local genomic instability. Instability of the SpTrf gene cluster was tested by 10 days of growth of Escherichia coli harboring bacterial artificial chromosome (BAC) clones of sea urchin genomic DNA with inserts containing SpTrf genes. After the growth period, the BAC DNA inserts were analyzed for size and SpTrf gene content. Clones with multiple SpTrf genes showed a variety of deletions, including loss of one, most, or all genes from the cluster. Alternatively, a BAC insert with a single SpTrf gene was stable. BAC insert instability is consistent with variations in the gene family composition among sea urchins, the types of SpTrf genes in the family, and a reduction in the gene copy number in single coelomocytes. Based on the sequence variability among SpTrf genes within and among sea urchins, local genomic instability of the family may be important for driving sequence diversity in this gene family that would be of benefit to sea urchins in their arms race with marine microbes.
IOS 1550747; IOS 1855747 National Science Foundation, na Cross Disciplinary Research Fund from the Columbian College of Arts and Sciences at George Washington University, na Wilber V. Harlan Graduate Scholarship, the Department of Biology , George Washington University, na Mortensen Award, the Department of Biology , George Washington University, na Dissertation Fellowship, the Columbian College of Arts and Sciences, George Washington University, 2208923 National Science Foundation
Figure 1. Graphical representation of BAC inserts that contain SpTrf gene locus 1, allele 1. (A) A graphical alignment of selected SpTrf genes showing the element pattern of each gene according to the repeat-based alignment [19]. Genes are composed of two exons; the first encodes the leader (L), and the second encodes the mature protein. All genes have a single, short intron (int) with several identifiable sequences that are indicated by the letters [19]. Exon 2 is a mosaic of elements (all of which are shown with numbers at the top) and is variable among the genes. Element 10 is labeled with different letters that indicate different sequences and define the element pattern. The horizontal black lines indicate missing elements. This figure is modified from Figure 6B in [34]. (B) The overlaps among the BAC inserts containing allele 1 from locus 1 are highlighted with the blue box. The black lines to the left and right of the blue box indicate the size of the flanking regions of the inserts that are not part of the gene cluster. This figure is modified from Figure 4A in [35].
Figure 2. BACs isolated from some colonies after 10 days of growth have small inserts. (A,C,E) Representative NotI digests of four BACs containing SpTrf genes in locus 1, allele 1 identify BACs with decreased insert sizes. BAC-con clones (C lanes, white arrows) that were grown overnight once show full-length inserts. The C lanes are followed by eight representative samples of BAC DNA isolated from single colonies after 10 days of growth. The colored arrows indicate BACs with small inserts. The first lane in (C) shows a PFGE lambda ladder (Bio-Rad Laboratories, Hercules, CA, USA). (B,D,F) PCR amplicons of genes in BAC inserts illustrate the genes in the cluster and identify the genes that are missing after 10 days of growth. Genes were amplified using primers specific for SpTrf genes (see Materials and Methods). The colored arrows in (A,C) correspond to the arrows in (B,D), respectively. Colored arrows indicate the BACs in which the gene copy number is different from the BAC-con clones (white arrows). The first or last lanes of the gels show the DNA ladder (Hi Lo DNA Standard, Fisher Scientific, Hampton, NH, USA). The legend for SpTrf gene amplicons shows the bands that correlate with individual genes based on sizes. SpTrf-A2 is the largest gene; the cluster of bands of intermediate size includes SpTrf-B8, -D1, and -E2 (three D1 genes result in the strongest bands in the center); and SpTrf-01 is the smallest. The marker is the Hi Lo DNA standard.
Figure 3. Locations of insert deletions are predicted by restriction digests. (A) XhoI/NotI and SacII/NotI virtual digests (https://nc3.neb.com/NEBcutter/ accessed on 18 May 2018) of full-length BAC insert sequences show the fragment sizes and on which fragments the SpTrf genes are located. (B) NotI double digests with XhoI or SacII show altered band sizes from BAC clones with small inserts and multiple SpTrf genes. BAC-51-con and BAC-52-con with full-length inserts show bands of expected size based on the virtual digests. The ladder is lambda DNA (monocot λ mix; New England Bio-Labs). (C–H) Maps of virtual digests predict the locations of the deleted regions. The maps are shown in linear format, even though the BAC DNA is circular. The areas in red and yellow indicate the predicted deletions based on results in (B). The SpTrf genes are indicated by colored dots based on the gene colors in Figure 1. The green segment indicates the pBACe3.6 vector. Restriction endonuclease sites in the BAC DNA are indicated as N, NotI; X, XhoI; S, SacII. (I) PCR is used to orient and verify the size change in BAC-51-15 (see C). Primers (pBACe3.6F or pBACe3.6R) that surround the vector and the R9 primer that is located within each gene (see red arrows in (C)) confirm a ~90 kb deletion in BAC-51-15 that results in a 4 kb amplicon. The gel image is edited to delete irrelevant lanes and bring the DNA standard (Hi Lo; Fisher Scientific) next to the lanes of interest.
Figure 4. Dot plots of sequenced BAC inserts identify deletions by comparisons to the full-length BAC insert sequences. Dot plots compare each full-length BAC insert to itself and to other BAC inserts that show deletions. The gene cluster maps of BAC-51 or BAC-52 are shown at the bottom of each dot plot with the location of each gene indicated by colored dots (based on the gene colors in Figure 1). Deletions identified by the dot plots are indicated by yellow highlights in the cluster maps and the number of nucleotides in each deletion is indicated. (A) BAC-51 vs. self. (B) BAC-52 vs. self. (C) BAC-51 vs. BAC-51-15. (D) BAC-52 vs. BAC-52-2b. (E) BAC-52 vs. BAC-52-con. (F) BAC-52 vs. BAC-52-con. (G) BAC-52 vs. BAC-52-4c.
Figure 5. BAC insert assemblies generate false deletions from low-quality sequencing reads. Sequence reads for each BAC are mapped onto reference sequences for BAC-51 (green maps) or BAC-52 (blue maps) that have been reported previously [35]. The lengths of the reference sequences for BAC-51 and BAC-52 are indicated and a ruler is included for every 20 kb. Mapping histograms for each BAC compared to the reference sequence indicate the depth of sequencing coverage and reads per position from the raw PacBio sequencing data. Higher peaks indicate greater coverage, while the absence of peaks indicates no sequence coverage. The colored bars within the histograms indicate nucleotide positions that have increased nucleotide variability across sequence reads. The height of each colored bar indicates the number of sequences for a specific nucleotide. Bar colors indicate nucleotides: red (T), orange (G), light green (A), and dark blue (C).
Figure 6. Many BAC inserts do not show SpTrf sequences by Southern blot. (A) BAC clones that did not amplify SpTrf sequences by PCR were digested with SalI and NotI, and the fragments were separated by gel electrophoresis, transferred to nylon filters, and evaluated with 32P-riboprobes. A subset of those BAC clones are shown. The yellow arrowheads indicate bands with SpTrf sequences that correspond to the bands in (B). The marker lanes (m) are Hi Lo DNA standards (Fisher Scientific). (B) The probes hybridize to three BAC inserts. The raw reads for BAC-42 and BAC-44 are available as BioSamples in GenBank, accession numbers SAMN39322606 and SAMN39322605, respectively. Preliminary sequence analysis of BAC-13 indicates that it includes a region of allele 2 for the SpTrf gene cluster in locus 1. Consequently, because this study focused on allele 1 of locus 1, long-read sequencing of the BAC-13 insert was not pursued. (C) BAC-7096 with six SpTrf genes (see Table 1, Figure 1B) [35,38] is the positive control for the Southern blot.
