scholarly journals Exon recognition and nucleocytoplasmic partitioning determine AMPD1 alternative transcript production.

1991 ◽  
Vol 11 (10) ◽  
pp. 5356-5363 ◽  
Author(s):  
I Mineo ◽  
E W Holmes
Keyword(s):  
Rna Processing ◽  
Donor Site ◽  
Alternative Transcript ◽  
Primary Transcript ◽  
Exon 2 ◽  
Mature Mrna ◽  
Cell Biol ◽  
Ampd1 Gene ◽  
Exon 1 ◽  
Intron 2

Two mature transcripts are produced from the rat AMP deaminase 1 (AMPD1) gene, one that retains exon 2 and one from which exon 2 has been removed. The ratio of these two transcripts is controlled by stage-specific and tissue-specific signals (I. Mineo, P. R. H. Clarke, R. L. Sabina, and E. W. Holmes, Mol. Cell. Biol. 10:5271-5278, 1990; R. L. Sabina, N. Ogasawara, and E. W. Holmes, Mol. Cell. Biol. 9:2244-2246, 1989). By using transfection studies with native, mutant, and chimeric minigene constructs, two steps in RNA processing that determine the ratio of these two transcripts have been identified. The first step is recognition of this exon in the primary transcript. The primary transcript is subject to alternative splicing in which exon 2 is either recognized and thereby included in the mature mRNA or is ignored and retained in a composite intron containing intron 1-exon 2-intron 2. The following properties of the primary transcript influence exon recognition. (i) Exon 2 is intrinsically difficult to recognize, possibly because of its small size (only 12 bases) and/or a suboptimal 5' donor site at the exon 2-intron 2 boundary. (ii) Intron 2 plays a permissive role in recognition of exon 2 because it is removed at a relatively slow rate, presumably because of the suboptimal polypyrimidine tract in the putative 3' branch site. The second step in RNA processing that influences the ratio of mature transcripts produced from the AMPD1 gene occurs subsequent to the ligation of exon 2 to exon 1. An RNA intermediate, composed of exon 1-exon 2-intron 2-exon 3, is produced in the first processing step, but it is variably retained in the nucleus. Retention of this intermediate in the nucleus is associated with accumulation of the mature mRNA containing exon 2, while cytoplasmic escape of this intermediate is reactions, exon recognition and nucleocytoplasmic partitioning, determine the relative abundance of alternative mRNAs derived from the AMPD1 gene.

Exon recognition and nucleocytoplasmic partitioning determine AMPD1 alternative transcript production

1991 ◽  
Vol 11 (10) ◽  
pp. 5356-5363
Author(s):  
I Mineo ◽  
E W Holmes
Keyword(s):  
Rna Processing ◽  
Donor Site ◽  
Alternative Transcript ◽  
Primary Transcript ◽  
Exon 2 ◽  
Mature Mrna ◽  
Cell Biol ◽  
Ampd1 Gene ◽  
Exon 1 ◽  
Intron 2

Two mature transcripts are produced from the rat AMP deaminase 1 (AMPD1) gene, one that retains exon 2 and one from which exon 2 has been removed. The ratio of these two transcripts is controlled by stage-specific and tissue-specific signals (I. Mineo, P. R. H. Clarke, R. L. Sabina, and E. W. Holmes, Mol. Cell. Biol. 10:5271-5278, 1990; R. L. Sabina, N. Ogasawara, and E. W. Holmes, Mol. Cell. Biol. 9:2244-2246, 1989). By using transfection studies with native, mutant, and chimeric minigene constructs, two steps in RNA processing that determine the ratio of these two transcripts have been identified. The first step is recognition of this exon in the primary transcript. The primary transcript is subject to alternative splicing in which exon 2 is either recognized and thereby included in the mature mRNA or is ignored and retained in a composite intron containing intron 1-exon 2-intron 2. The following properties of the primary transcript influence exon recognition. (i) Exon 2 is intrinsically difficult to recognize, possibly because of its small size (only 12 bases) and/or a suboptimal 5' donor site at the exon 2-intron 2 boundary. (ii) Intron 2 plays a permissive role in recognition of exon 2 because it is removed at a relatively slow rate, presumably because of the suboptimal polypyrimidine tract in the putative 3' branch site. The second step in RNA processing that influences the ratio of mature transcripts produced from the AMPD1 gene occurs subsequent to the ligation of exon 2 to exon 1. An RNA intermediate, composed of exon 1-exon 2-intron 2-exon 3, is produced in the first processing step, but it is variably retained in the nucleus. Retention of this intermediate in the nucleus is associated with accumulation of the mature mRNA containing exon 2, while cytoplasmic escape of this intermediate is reactions, exon recognition and nucleocytoplasmic partitioning, determine the relative abundance of alternative mRNAs derived from the AMPD1 gene.

scholarly journals A novel pathway for alternative splicing: identification of an RNA intermediate that generates an alternative 5' splice donor site not present in the primary transcript of AMPD1.

1990 ◽  
Vol 10 (10) ◽  
pp. 5271-5278 ◽  
Author(s):  
I Mineo ◽  
P R Clarke ◽  
R L Sabina ◽  
E W Holmes
Keyword(s):  
Alternative Splicing ◽  
Donor Site ◽  
Specific Pattern ◽  
Splice Donor Site ◽  
Splice Donor ◽  
Primary Transcript ◽  
Exon 2 ◽  
Tissue Specific ◽  
Exon 1 ◽  
Alternatively Spliced

AMP deaminase (AMPD) is a central enzyme in eucaryotic energy metabolism, and tissue-specific as well as stage-specific isoforms are found in many vertebrates. This study demonstrates the AMPD1 gene product in rat is alternatively spliced. The second exon, a 12-base miniexon, was found to be excluded or included in a tissue-specific and stage-specific pattern. This example of cassette splicing utilizes a unique pathway through an RNA intermediate that generates an alternative 5' splice donor site at the point where exon 2 is ligated to exon 1. In the analogous intermediate of human AMPD1, the potential 5' splice donor site created at the boundary of exon 1 and exon 2 was a poor substrate for splicing because of differences in exon 2 sequences, and human AMPD1 was not alternatively spliced. These results demonstrate that in some cases alternative splicing may proceed through an RNA intermediate that generates an alternative splice donor site not present in the primary transcript. Discrimination between alternative 5' splice donor sites in the RNA intermediate of AMPD1 is apparently controlled by tissue-specific and stage-specific signals.

A novel pathway for alternative splicing: identification of an RNA intermediate that generates an alternative 5' splice donor site not present in the primary transcript of AMPD1

1990 ◽  
Vol 10 (10) ◽  
pp. 5271-5278
Author(s):  
I Mineo ◽  
P R Clarke ◽  
R L Sabina ◽  
E W Holmes
Keyword(s):  
Alternative Splicing ◽  
Donor Site ◽  
Specific Pattern ◽  
Splice Donor Site ◽  
Splice Donor ◽  
Primary Transcript ◽  
Exon 2 ◽  
Tissue Specific ◽  
Exon 1 ◽  
Alternatively Spliced

AMP deaminase (AMPD) is a central enzyme in eucaryotic energy metabolism, and tissue-specific as well as stage-specific isoforms are found in many vertebrates. This study demonstrates the AMPD1 gene product in rat is alternatively spliced. The second exon, a 12-base miniexon, was found to be excluded or included in a tissue-specific and stage-specific pattern. This example of cassette splicing utilizes a unique pathway through an RNA intermediate that generates an alternative 5' splice donor site at the point where exon 2 is ligated to exon 1. In the analogous intermediate of human AMPD1, the potential 5' splice donor site created at the boundary of exon 1 and exon 2 was a poor substrate for splicing because of differences in exon 2 sequences, and human AMPD1 was not alternatively spliced. These results demonstrate that in some cases alternative splicing may proceed through an RNA intermediate that generates an alternative splice donor site not present in the primary transcript. Discrimination between alternative 5' splice donor sites in the RNA intermediate of AMPD1 is apparently controlled by tissue-specific and stage-specific signals.

Identification of Three Novel Substitutions in the Erythroid Promoter of the HMBS Gene: A New Erythroid Variant of Human Acute Intermittent Porphyria?

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4574-4574
Author(s):  
Elena Di Pierro ◽  
Valeria Besana ◽  
Valentina Brancaleoni ◽  
Dario Tavazzi ◽  
Maria Domenica Cappellini
Keyword(s):  
Acute Intermittent Porphyria ◽  
Normal Subjects ◽  
Normal Function ◽  
Inherited Disorders ◽  
Exon 2 ◽  
Molecular Defects ◽  
Exon 1 ◽  
Classic Form ◽  
Partial Deficiency ◽  
Intron 2

Abstract Porphyrias are inherited disorders in the biosynthesis of heme. Acute intermittent porphyria (AIP), the most common form, is autosomal dominant and it is characterized by recurrent attacks of abdominal pain, gastrointestinal dysfunction, and neurologic disturbances. AIP is caused by molecular defects in the hydroxymethylbilane synthase gene (HMBS) causing a partial deficiency of the third enzyme of the heme biosynthetic pathway. This gene maps to chromosome 11q23.2 with a total of 15 exons. Two distinct promoters direct the synthesis of housekeeping and erythroid specific mRNAs by alternative splicing. The housekeeping promoter is located upstream the exon 1 while the erythroid promoter include a portion of intron 1, the exon 2 and the intron 2. The exon 1 and the exons 3 to 15 generate the housekeeping mRNA while the exons 2 to 15 generate the erythroid mRNA giving rise to the housekeeping HMBS isoform and to the erythroid HMBS isoform respectively. In the classic form of AIP, both the housekeeping and the erythroid HMBS isoforms are deficient since the must common molecular defects involve the common region of the gene from exon 3 to exon 15. Approximately 5% of AIP patients have normal levels of the erythroid HMBS isoform and defects in the housekeeping promoter or in the exon 1 which is specific for the housekeeping HMBS isoform, causing the ‘non-erythroid variant’ of AIP. So far no mutations are known in the erythroid promoter. In this study we searched for molecular defects in HMBS gene in three Italian patients with typical clinical and biochemical signs of AIP. The diagnosis was based on elevated urinary excretion of porphyrinic precursors and reduced erythrocyte HMBS activity. The entire HMBS gene has been amplified by PCR and submitted to direct automated sequencing: no mutations known as responsible for classic form of AIP have been identified. However, each of three patients had a substitutions in the erythroid promoter of HMBS gene that could justify the reduced erythrocyte HMBS activity: c.34–115 C>A, c.34–126 G>C and c.34–156G>A. In order to establish if these substitutions were polymorphisms, more than 200 alleles from Italian normal subjects were sequenced and none of them revealed the substitutions. These three mutations, located specifically in the intron 2, could affect the normal splicing of exon 3 causing an abnormal mRNA, to give rise to the classical form of AIP. The mRNA analysis, however, didn’t reveal any abnormal mRNA. Moreover real time experiments in the lymphocytes showed a 100% expression of housekeeping HMBS mRNA excluding the non-sense mediated decay mechanism. These data suggest that these mutations could affect the normal function of the erythroid promoter only, causing an erythroid variant of human acute intermittent porphyria. Further expression studies are in progress.

scholarly journals Molecular characterization of novel germline deletions affecting SDHD and SDHC in pheochromocytoma and paraganglioma patients

2009 ◽  
Vol 16 (3) ◽  
pp. 929-937 ◽  
Author(s):  
Jean-Pierre Bayley ◽  
Marjan M Weiss ◽  
Anneliese Grimbergen ◽  
Bernadette T J van Brussel ◽  
Frederik J Hes ◽  
...  
Keyword(s):  
Nonsense Mutations ◽  
Gene Deletions ◽  
Exon 2 ◽  
Gene Insertion ◽  
Large Deletions ◽  
Exon 1 ◽  
Long Range Pcr ◽  
Intron 2 ◽  
Dependent Probe

A major cause of paraganglioma and pheochromocytoma is germline mutation of the tumor suppressor genes SDHB, SDHC, and SDHD, encoding subunits of succinate dehydrogenase (SDH). While many SDH missense/nonsense mutations have been identified, few large deletions have been described. We performed multiplex ligation-dependent probe amplification deletion analysis in 126 point mutation-negative patients, and here we describe four novel deletions of SDHD and SDHC. Long-range PCR was used for the fine mapping of deletions. One patient had a 10 kb AluSg–AluSx-mediated deletion including SDHD exons 1 and 2, the entire TIMM8B gene, and deletion of exons of C11orf57. A second patient had a deletion of SDHD exons 1 and 2 and exon 1 of the TIMM8B gene. A third patient showed a deletion of exon 2 of SDHD, together with a 235 bp MIRb–Tensin gene insertion. In a fourth patient, a deletion of exons 5 and 6 of the SDHC gene was found, only the second SDHC deletion currently known. The deletions of the TIMM8B and C11orf57 genes are the first to be described, but do not appear to result in an additional phenotype in these patients. Four of the eight breakpoints occurred in Alu sequences and all three SDHD deletions showed an intron 2 breakpoint. This study underlines the fact that clinically relevant deletions may encompass neighboring genes, with the potential to modify phenotype. Gene deletions of SDHD and SDHC represent a substantial proportion of all mutations, and must be considered in paraganglioma patients shown to be negative for mutations by sequencing.

scholarly journals Multiple transcripts of the CYP21 gene are generated by the mutation of the splicing donor site in intron 2 from GT to AT in 21-hydroxylase deficiency

2001 ◽  
Vol 171 (3) ◽  
pp. 397-402 ◽  
Author(s):  
HH Lee ◽  
SF Chang
Keyword(s):  
Donor Site ◽  
Splice Acceptor Site ◽  
Splice Donor Site ◽  
Acceptor Site ◽  
Site Mutation ◽  
Hydroxylase Deficiency ◽  
Exon 2 ◽  
Eukaryotic Genes ◽  
21 Hydroxylase Deficiency ◽  
Intron 2

Maturation of primary RNA transcripts of eukaryotic genes often involves the removal of introns and joining of exons. The fidelity of RNA splicing is dependent on the identity of the nucleotide (nt) sequences at exon/intron boundaries. Most importantly, the highly conserved intronic 5'GT and 3'AG sequences are essential for correct splicing. Substitution of GT by any other nt leads to incomplete mRNA and a disruption of protein structure. We describe here the results of our transfection experiments in COS-1 cells with a CYP21 genomic construct that contained an IVS 2+1G-->A mutation. Analysis of the transcripts by RT-PCR revealed that two different transcripts were generated by this mutant genome. In all the splicing products, we found that the entire exon 2 was deleted. Surprisingly, 30% of the transcripts from this mutant CYP21 genome were accompanied by an inclusion of 3' intron 2 sequences due to the use of a different splice acceptor site. This is the first report of the molecular characterization of a splice donor site mutation in CYP21 via transcription in COS-1 cells.

Mechanisms That Link Promoter Choice with Downstream Alternative Splicing in the Erythroid Protein 4.1R Gene.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1562-1562
Author(s):  
Marilyn Parra ◽  
Jeff Tan ◽  
Narla Mohandas ◽  
John G. Conboy
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Rna Processing ◽  
Regulatory Element ◽  
Donor Site ◽  
Splice Acceptor Site ◽  
Acceptor Site ◽  
Splice Acceptor ◽  
Exon 2 ◽  
Protein 4.1R

Abstract The protein 4.1R gene is a large transcription unit (240kb) that utilizes complex RNA processing pathways to encode distinct protein isoforms, both during erythropoiesis and also in nonerythroid cells. Proper regulation of these pathways is essential for stage-specific synthesis of the 80-kDa isoforms of 4.1R protein during terminal erythroid differentiation. The 5′ region of the gene contains multiple alternative first exons that map far upstream of the coding exons, and we have shown previously that promoter choice is coupled to alternative splicing decisions 100kb downstream in exon 2′/2. Transcripts that initiate at exon 1A predominate in late stages of erythropoiesis and splice only to a weak internal 3′ splice acceptor site in exon 2, skipping translation start site AUG1 and ensuring proper translation initiation at AUG2 in exon 4 for synthesis of the 80-kDa isoforms. In contrast, 4.1 transcripts initiated at exons 1B or 1C exclusively splice to the strong first 3′ splice acceptor site at exon 2′ to include AUG1 and encode a higher molecular weight 135-kDa isoform known to interact with different affinity to major erythroid membrane proteins in earlier stages of erythropoiesis. Our studies show that this linkage between transcription and splicing is (a) cell type independent; (b) conserved in the 4.1R gene from fish to man; and (c) conserved in the paralogous 4.1B gene. Our recent functional studies suggest that a novel re-splicing mechanism, reminiscent of recursive splicing of large introns previously described in the Drosophila ubx gene, may couple promoter choice with downstream splicing in the 4.1R gene. Using minigenes that reproduce the differential splicing patterns in transfected mammalian cells, we have shown that accurate splicing of exon 1A requires a unique downstream regulatory element. This element maps several kilobases downstream of exon 1A and is conserved among mammals. Analysis of wild type and mutated minigenes suggests a two step splicing model in which this element behaves as a temporary “intra-exon” that is present in a splicing intermediate but eliminated from the mature mRNA. According to this model, the regulatory element behaves as an exon in the first step as its consensus 5′ donor site splices to the strong 3′ splice site of exon 2′, removing this splice site pair and joining the intra-exon directly to exon 2′. In the second step, the juxtaposed region of the intra-exon then behaves as an intron, contributing to the activation of the weak internal splice acceptor at exon 2. This second splicing event joins exon 1A to exon 2, thus deleting the intra-exon, the 2′ region (and AUG1) and generating a mature 5′ end capable of encoding 80-kDa 4.1R. Importantly, pre-mRNA constructs that lack the intra-exon, or have a mutated intra-exon 5′ splice donor site, are uncoupled and exhibit inappropriate splicing of exon 1A to the first acceptor site at exon 2′. In support of the generality of this model, we have identified a candidate intra-exon with similar sequence properties in the long 5′ region of the human 4.1B gene, and have demonstrated that this element successfully rescues proper splicing of 4.1R exon 1A in our minigenes. Detailed molecular analysis is under way to identify the specific cis and trans elements required to effect this unusual, long-distance coupling between RNA processing events which have implications for detailed mechanistic understanding of membrane assembly during erythropoiesis.

scholarly journals 5′ Region Large Genomic Rearrangements in the BRCA1 Gene in French Families: Identification of a Tandem Triplication and Nine Distinct Deletions with Five Recurrent Breakpoints

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3171
Author(s):  
Sandrine M. Caputo ◽  
Dominique Telly ◽  
Adrien Briaux ◽  
Julie Sesen ◽  
Maurizio Ceppi ◽  
...  
Keyword(s):  
Brca1 Gene ◽  
Genomic Rearrangements ◽  
Comparative Genomic ◽  
Homologous Region ◽  
Exon 2 ◽  
Large Genomic Rearrangements ◽  
Causality Score ◽  
Frequent Site ◽  
First Time ◽  
Intron 2

Background: Large genomic rearrangements (LGR) in BRCA1 consisting of deletions/duplications of one or several exons have been found throughout the gene with a large proportion occurring in the 5′ region from the promoter to exon 2. The aim of this study was to better characterize those LGR in French high-risk breast/ovarian cancer families. Methods: DNA from 20 families with one apparent duplication and nine deletions was analyzed with a dedicated comparative genomic hybridization (CGH) array, high-resolution BRCA1 Genomic Morse Codes analysis and Sanger sequencing. Results: The apparent duplication was in fact a tandem triplication of exons 1 and 2 and part of intron 2 of BRCA1, fully characterized here for the first time. We calculated a causality score with the multifactorial model from data obtained from six families, classifying this variant as benign. Among the nine deletions detected in this region, eight have never been identified. The breakpoints fell in six recurrent regions and could confirm some specific conformation of the chromatin. Conclusions: Taken together, our results firmly establish that the BRCA1 5′ region is a frequent site of different LGRs and highlight the importance of the segmental duplication and Alu sequences, particularly the very high homologous region, in the mechanism of a recombination event. This also confirmed that those events are not systematically deleterious.

scholarly journals Transcriptional Pausing and Activation at Exons-1 and -2, Respectively, Mediate the MGMT Gene Expression in Human Glioblastoma Cells

Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 888
Author(s):  
Mohammed A. Ibrahim Al-Obaide ◽  
Kalkunte S. Srivenugopal
Keyword(s):  
Dna Methyltransferase ◽  
Glioblastoma Cells ◽  
Rt Pcr ◽  
Repair Gene ◽  
Exon 2 ◽  
Mgmt Gene ◽  
Dna Repair Gene ◽  
Exon 1 ◽  
Transcriptional Pausing ◽  
Transcription Assays

Background: The therapeutically important DNA repair gene O6-methylguanine DNA methyltransferase (MGMT) is silenced by promoter methylation in human brain cancers. The co-players/regulators associated with this process and the subsequent progression of MGMT gene transcription beyond the non-coding exon 1 are unknown. As a follow-up to our recent finding of a predicted second promoter mapped proximal to the exon 2 [Int. J. Mol. Sci.2021, 22(5), 2492], we addressed its significance in MGMT transcription. Methods: RT-PCR, RT q-PCR, and nuclear run-on transcription assays were performed to compare and contrast the transcription rates of exon 1 and exon 2 of the MGMT gene in glioblastoma cells. Results: Bioinformatic characterization of the predicted MGMT exon 2 promoter showed several consensus TATA box and INR motifs and the absence of CpG islands in contrast to the established TATA-less, CpG-rich, and GAF-bindable exon 1 promoter. RT-PCR showed very weak MGMT-E1 expression in MGMT-proficient SF188 and T98G GBM cells, compared to active transcription of MGMT-E2. In the MGMT-deficient SNB-19 cells, the expression of both exons remained weak. The RT q-PCR revealed that MGMT-E2 and MGMT-E5 expression was about 80- to 175-fold higher than that of E1 in SF188 and T98G cells. Nuclear run-on transcription assays using bromo-uridine immunocapture followed by RT q-PCR confirmed the exceptionally lower and higher transcription rates for MGMT-E1 and MGMT-E2, respectively. Conclusions: The results provide the first evidence for transcriptional pausing at the promoter 1- and non-coding exon 1 junction of the human MGMT gene and its activation/elongation through the protein-coding exons 2 through 5, possibly mediated by a second promoter. The findings offer novel insight into the regulation of MGMT transcription in glioma and other cancer types.

scholarly journals Plasmodium vivax Cysteine-Rich Protective Antigen Polymorphism at Exon-1 Shows Recombination and Signatures of Balancing Selection

Genes ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 29
Author(s):  
Lilia González-Cerón ◽  
José Cebrián-Carmona ◽  
Concepción M. Mesa-Valle ◽  
Federico García-Maroto ◽  
Frida Santillán-Valenzuela ◽  
...  
Keyword(s):  
Amino Acid ◽  
B Cell ◽  
Plasmodium Vivax ◽  
Nucleotide Diversity ◽  
Protective Antigen ◽  
Balancing Selection ◽  
Southern Mexico ◽  
Exon 2 ◽  
Exon 1 ◽  
Tajima’S D

Plasmodium vivax Cysteine-Rich Protective Antigen (CyRPA) is a merozoite protein participating in the parasite invasion of human reticulocytes. During natural P. vivax infection, antibody responses against PvCyRPA have been detected. In children, low anti-CyRPA antibody titers correlated with clinical protection, which suggests this protein as a potential vaccine candidate. This work analyzed the genetic and amino acid diversity of pvcyrpa in Mexican and global parasites. Consensus coding sequences of pvcyrpa were obtained from seven isolates. Other sequences were extracted from a repository. Maximum likelihood phylogenetic trees, genetic diversity parameters, linkage disequilibrium (LD), and neutrality tests were analyzed, and the potential amino acid polymorphism participation in B-cell epitopes was investigated. In 22 sequences from Southern Mexico, two synonymous and 21 nonsynonymous mutations defined nine private haplotypes. These parasites had the highest LD-R2 index and the lowest nucleotide diversity compared to isolates from South America or Asia. The nucleotide diversity and Tajima’s D values varied across the coding gene. The exon-1 sequence had greater diversity and Rm values than those of exon-2. Exon-1 had significant positive values for Tajima’s D, β-α values, and for the Z (HA: dN > dS) and MK tests. These patterns were similar for parasites of different origin. The polymorphic amino acid residues at PvCyRPA resembled the conformational B-cell peptides reported in PfCyRPA. Diversity at pvcyrpa exon-1 is caused by mutation and recombination. This seems to be maintained by balancing selection, likely due to selective immune pressure, all of which merit further study.

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