Single Base Insertion Research Articles

Identification and characterization of hydroxymethylbilane synthase mutations causing acute intermittent porphyria: Evidence for an ancestral founder of the common G111R mutation

Acute intermittent porphyria (AIP), the most common hepatic porphyria, results from the half-normal activity of hydroxymethylbilane synthase (HMB-synthase; EC 4.3.1.8), the third enzyme in the heme biosynthetic pathway. Because life-threatening acute neurologic attacks of this autosomal dominant disease are triggered by various ecogenic factors (e.g., certain drugs, hormones, alcohol, and starvation), efforts have been directed to identify and counsel presymptomatic heterozygotes in affected families to avoid the precipitating factors. Thus, to determine the nature of the mutations causing AIP in 26 unrelated enzyme-confirmed patients from Argentina, a long-range polymerase chain reaction method was developed to amplify the entire 10-kb gene in two fragments for efficient cycle sequencing and mutation detection. Eight new mutations were identified including two missense mutations (Q34P and G335S), four small deletions (728delCT, 815delAGGA, 948delA, and 985del12), a single base insertion (666insA), and a splice site mutation (IVS12(+1)). In addition, five previously reported mutations (G111R, R173W, Q204X, R201W, and 913insC) were detected. Notably, G111R was identified in 12 of the 26 (46%) presumably unrelated propositi; however, haplotype analysis with intragenic and flanking markers indicated an ancestral founder. Expression of the two new missense mutations (Q34P and G335S) in f1 E. coli resulted in 2.5% or less of the normal expressed enzyme, confirming their defective function. Thus, eight new and five previously reported HMB-synthase mutations, including a common lesion, were detected, permitting accurate identification and counseling of presymptomatic carriers in these 26 unrelated Argentinean AIP families with this dominant porphyria.

NalB-type mutations causing the overexpression of the MexAB-OprM efflux pump are located in the mexR gene of the Pseudomonas aeruginosa chromosome

Two mutations, one at the nalB and the other at the mexR locus, in the Pseudomonas aeruginosa chromosome are known to cause overexpression of the MexAB-OprM efflux pump. Based on the following results, we concluded that nalB is the mutation that has occurred within the mexR gene of the P. aeruginosa chromosome. (i) Nucleotide sequencing of the mex operon upstream region of 21 independent nalB-type mutants including the original nalB9 revealed that all the mutations were located within the mexR gene. The mutations were classified into three different groups and nine types including single base substitutions, single base deletions and base insertions. (ii) Substitution of the mutant mexR with the wild-type mexR and replacement of the wild-type mexR with a defined mexR mutation resulted in the expression of wild-type and nalB-type MexAB-OprM respectively, which was confirmed by testing the antibiotic susceptibility and β-galactosidase activity of the mexA-lacZ translational fusion.

X-linked dominant Charcot–Marie–Tooth neuropathy: clinical, electrophysiological, and morphological phenotype in four families with different connexin32 mutations

The sensorimotor neuropathy of the Charcot-Marie-Tooth type (CMT) is the most common hereditary disorder of the peripheral nervous system. The X-linked dominant form of CMT (CMTX) is associated with mutations in the gene for the gap junction protein connexin32. We examined four CMTX pedigrees two of which had potentially novel mutations in the only coding exon of connexin32. One previously unreported missense mutation, Ala39Val, was found in a family displaying a CMT phenotype with additional upper limb postural tremor reminiscent of a Roussy-Lévy syndrome. A novel single base insertion, 679insT, is among the first mutations found in the fourth transmembrane domain of connexin32. Frameshift and premature stop of translation are supposed to result in a non-functional carboxy-terminus. Two further families had the known missense mutations Arg15Trp and Arg22Gln. Several female carriers were found normal on clinical presentation, however, the genotype was paralleled by decreased nerve conduction velocities (NCV) and slowed central conduction of brain stem auditory evoked responses (BAER). Median motor NCVs showed mild (in women) to intermediate (in males) reduction, indicating a peripheral neuropathy with a predominating axonal component. Nerve biopsy findings were consistent with the electrophysiological data showing a marked loss of large myelinated fibres and clusters of regenerating axons. Electron microscopy revealed various alterations of the axoglial attachment zone. This suggests defective axon-Schwann cell interactions which may induce the axonopathy in CMTX.

DbSNP—Database for Single Nucleotide Polymorphisms and Other Classes of Minor Genetic Variation

A key aspect of research in genetics is associating sequence variations with heritable phenotypes. The most common variations are single nucleotide polymorphisms (SNPs), which occur approximately once every 500–1000 bases in a large sample of aligned human sequence. Because SNPs are expected to facilitate large-scale association genetics studies, there has recently been great interest in SNP discovery and detection. In collaboration with the National Human Genome Research Institute (NHGRI), the National Center for Biotechnology Information (NCBI) has established the dbSNP database (http://www.ncbi.nlm. nih.gov/SNP) to serve as a central repository for molecular variation. Designed to serve as a general catalog of molecular variation to supplement GenBank (Benson et al. 1999) database submissions can include a broad range of molecular polymorphisms: single base nucleotide substitutions, short deletion and insertion polymorphisms, microsatellite markers, and polymorphic insertion elements such as retrotransposons. Although the name dbSNP is a slight misnomer given the variations represented, SNP polymorphisms are the largest class of variation in the database, and the name dbSNP, selected at the request of NHGRI, reflects this fact. For the sake of brevity, we elected to use the term SNP as a shorthand for “variation” in the database notation and documentation (http://www.ncbi.nlm.nih.gov/ SNP/get_html.cgi?whichHtml=how_to_ submit). Thus terms used in the documentation like “submitted SNP” or “reference SNP” refer to all classes of variation in the database and should be regarded as meaning “a submitted report of variation” and “a reference report of variation.” Furthermore, it should be noted that in serving its role as the variation complement to GenBank, dbSNP does not restrict submissions to only neutral polymorphisms. Submissions are welcome on all classes of simple molecular variation, including those that cause rare clinical phenotypes. Submissions to dbSNP come from a variety of sources including individual laboratories, collaborative polymorphism discovery efforts, large-scale genome sequencing centers, and private industry. The data collected range from the tightly focused characterization of particular genes to broadly sampled levels of variation from random genomic sequence. The distribution of reported marker density across the genome is thus expected to be mixed, with an expected minimum density of 1/3000 bases in regions of random genomic sequence, and local regions of higher density around well-characterized genes. Each variation submitted to dbSNP must have an identifier provided by the submitter (called a “local” identifier by dbSNP), and each is issued a unique identifier, formatted as an integer prefixed with ss (for submitted SNP), for example, ss334. An ss number is thus permanently associated with the submitter’s identifier, and it can be treated as a formal accession number by the scientific publishing community.

Allelic diversity of the human plasma alpha(1,3)fucosyltransferase gene (FUT6).

The 1080-bp coding region of the human plasma alpha(1,3)fucosyltransferase gene (FUT6) was sequenced in a total of 161 individuals (322 chromosomes) drawn from three populations, involving 56 Africans (Xhosa), 52 European-Africans of South Africa, and 53 Japanese. In addition to six reported base substitutions, eleven new base substitutions and a single base insertion were found in the coding region of the FUT6. Eleven functional and four null alleles were encountered, of which 10 alleles were novel alleles identified in this study. Two null alleles have been identified previously, whereas two novel null alleles, which contained a single base (cytosine) insertion at nucleotide 499, were found in a Xhosa population. The allelic distributions of FUT6 were different among these three populations. The heterozygosity of FUT6 was 0.860, 0.699, and 0.632, in Xhosa, European-African (South Africa), and in Japanese populations, respectively. The extensive DNA sequence diversity of the FUT6 may be suitable for application as a tool in genetic studies for modern human evolution.

Identification of Mutations in the Repeated Part of the Autosomal Dominant Polycystic Kidney Disease Type 1 Gene, PKD1, by Long-Range PCR

We have used long-range PCR to identify mutations in the duplicated part of the PKD1 gene. By means of a PKD1-specific primer in intron 1, an approximately 13.6-kb PCR product that includes exons 2-15 of the PKD1 gene has been used to search for mutations, by direct sequence analysis. This region contains the majority of the predicted extracellular domains of the PKD1-gene product, polycystin, including the 16 novel PKD domains that have similarity to immunoglobulin-like domains found in many cell-adhesion molecules and cell-surface receptors. Direct sequence analysis of exons encoding all the 16 PKD domains was performed on PCR products from a group of 24 unrelated patients with autosomal dominant polycystic kidney disease (ADPKD [MIM 173900]). Seven novel mutations were found in a screening of 42% of the PKD1-coding region in each patient, representing a 29% detection rate; these mutations included two deletions (one of 3 kb and the other of 28 bp), one single-base insertion, and four nucleotide substitutions (one splice site, one nonsense, and two missense). Five of these mutations would be predicted to cause a prematurely truncated protein. Two coding and 18 silent polymorphisms were also found. When, for the PKD1 gene, this method is coupled with existing mutation-detection methods, virtually the whole of this large, complex gene can now be screened for mutations.

A new variant CYP2D6 allele (CYP2D6*21) with a single base insertion in exon 5 in a Japanese population associated with a poor metabolizer phenotype.

Two poor metabolizer individuals of debrisoquine were identified among 215 healthy Japanese by a phenotyping test. Analysis of the CYP2D6 gene from one of two poor metabolizers, who was not homozygous for the previously described CYP2D6 variant alleles (CYP2D6*3, CYP2D6*4, CYP2D6*5 and CYP2D6*18), showed that this individual was heterozygous for a new allele, CYP2D6/C8 (CYP2D6*21). CYP2D6*21 had a single cytosine insertion at position 2661 in exon 5. This cytosine insertion generated a stop codon at the 17 bp downstream of this insertion site. A method to detect this allele was established with an allele specific-polymerase chain reaction. This method showed that another one of two poor metabolizers also possessed CYP2D6*21 allele heterozygously. In 318 healthy Japanese, five individuals carried this allele, heterozygously (0.81%, 5/636 chromosomes). Based on the present and our previous data, the poor metabolizer frequency in Japanese was estimated to be 0.39%, which accounted for approximately 45% of the individuals phenotyped as poor metabolizers by in-vivo tests.

Genotypic, immunophenotypic and clinical features of Thai patients with paroxysmal nocturnal haemoglobinuria

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haematological disorder characterized by complement-mediated haemolytic anaemia caused by deficiency of glycosylphosphatidylinositol (GPI) anchored proteins. Somatic mutation of an X-linked gene, PIG-A, is responsible for the defect in biosynthesis of GPI-anchor. It appears that frequency of PNH differs geographically, and seems to be more frequent in some Asian countries, such as Thailand and China. We studied a group of 34 Thai patients with PNH to see whether the somatic mutations in PIG-A, extent of deficiency of GPI-anchored proteins (complete or partial) and complication with aplastic anaemia among Thai patients are different from those in other regions. We determined 37 PIG-A mutations in 33 patients (10 base substitutions, 14 single-base deletions, five multiple-base deletions, three single-base insertions, two multiple base insertions and three others) which were found to be similar to those found in European, American and Japanese patients. Most patients had cells with a complete deficiency of CD59 (type III cells), whereas 19% and 33% of the patients with reliable data for CD59 expression had partially deficient granulocytes and erythrocytes (type II cells), respectively. Most mutations resulted in a complete loss of function of PIG-A in accordance with the prevalent PNH III phenotype. 19 patients (51%) had aplastic anaemia; their PIG-A mutations were not different from those without pre-existing aplastic anaemia. These characteristics of Thai patients are similar to patients from other regions. There was some negative correlation between mean basal Hb concentration and percentage of CD59-negative granulocytes (r = -0. 374; P = 0.0476). In addition, patients with severe anaemia (basal Hb <7 g/dl) had a significantly higher percentage of affected granulocytes than those with mild anaemia (88.5 +/- 9.4 v 64.9 +/- 25.9; P = 0.01). The data suggest that the severity of anaemia in PNH depends partly on the size of the PNH clone.

Identification and quantification of somatic mosaicism for a point mutation in a Duchenne muscular dystrophy family

Relatively few point mutations have been found in the dystrophin gene and of these only two have been associated with mosaicism. A single base insertion has been identified and quantified...

CD59-deficient blood cells and PIG-A gene abnormalities in Japanese patients with aplastic anaemia.

Patients with aplastic anaemia (AA) frequently develop paroxysmal nocturnal haemoglobinuria (PNH) as a late complication. We investigated the frequency of the development of PNH features including a glycosyl phosphatidylinositol (GPI) anchoring defect in 73 Japanese patients with AA. A deficient expression of CD59 was found on erythrocytes and/or granulocytes in 21/73 (28.8%) of the patients. A Ham/sugar water test was positive in 13/21 patients. We also examined mutations of the PIG-A gene in 11 patients with CD59 deficiency. A heteroduplex analysis detected PIG-A gene abnormality in 10/11 patients tested. Nucleotide sequencing was performed in six patients and identified eight mutations including three mutations in one patient. The mutations of the PIG-A gene were all different and included two single-base insertions, one single-base deletion, two two-base deletions, and one each of eight-base insertion and nine- and ten-base deletions. All mutations but one caused frameshifts. Our findings indicate that a high proportion of Japanese patients with severe AA have a GPI-anchoring defect and that the PIG-A gene is mutated in the AA patients who had a GPI deficiency. We found no significant difference in the pattern of the PIG-A gene mutation between the AA patients with a GPI deficiency and those with de novo PNH.

Chloride channel CLCN5 mutations in Japanese children with familial idiopathic low molecular weight proteinuria

Familial idiopathic low molecular weight proteinuria (FILMWP) is a renal proximal tubulopathy characterized by mild proteinuria consisting of low molecular weight proteinuria and relatively conserved renal function in young patients, but without rickets. Mutations in the renal chloride channel CLCN5 gene have been reported in three disorders of hypercalciuric nephrolithiasis and in FILMWP. To assess the relationship between molecular defects and phenotypic variations, we analyzed the CLCN5 gene from three additional Japanese families with FILMWP using single-strand conformation polymorphism and sequencing. We identified three mutations: a single base insertion at codon 514; a single base deletion at codon 116; and a nonsense mutation, R704X. The R704X mutation is identical to that found in X-linked recessive nephrolithiasis, but there was no renal failure in our patient. The first two mutations caused a shift in the reading frame, and all introduced a premature stop codon, resulting in synthesis of truncated CLC-5 proteins that lacked 220 (29%), 610 (82%), and 43 (6%) amino acids, respectively. These mutations were demonstrated to cosegregate with the disease in each of the three families. We conclude that the CLCN5 gene is responsible for the renal proximal tubulopathy in many Japanese families and suggest that molecular defects, environmental factors, or other modifying genes may account for the different phenotypes.

DNA sequence analysis of hprt mutants persisting in peripheral blood of cynomolgus monkeys more than two years after ENU treatment

We have been studying in vivo mutagenesis at the hypoxanthine phosphoribosyl transferase (hprt) locus in cynomolgus monkey T-lymphocytes. This primate model allows us to study mutations and their kinetics under well-controlled conditions. Previously, we reported mutations detected at various times after intraperitoneal treatment with ethylnitrosourea (ENU, 77 mg/kg). At 832 days after that first treatment, the monkey received a second dose of 77 mg/kg ENU. Up to 1,331 days after the second treatment, the T-cell mutant frequency (44.2 × 10−6) was still 26-fold higher than background (1.7 × 10−6), suggesting that mutants persisted in the peripheral blood. Mutant clones from Days 974, 1,164, and 1,311 after the second treatment were selected in thioguanine. Hprt cDNA was prepared from a cell lysate, PCR-amplified, and sequenced. Of 45 mutants, 30 yielded PCR product and 26 were sequenced. Base substitutions were found in 21 (81%) of the 26 mutants and consisted of one G:C → A:T and five A:T → G:C transitions, one G:C → C:G, eight A:T → T:A, and six A:T → C:G transversions. Therefore, most base substitutions occurred at A:T basepairs, characteristic of ENU-induced mutations in vivo, and were detected up to 3.6 years after the second treatment. Deletions of exons 2 and 3 occurred in two mutants and exon 7 was deleted in one mutant. There were two insertion mutants: one was a single base insertion and the other contained an insertion of 277 basepairs which was nearly identical to a simian retroviral sequence. Environ. Mol. Mutagen. 33:42–48, 1999 © 1999 Wiley-Liss, Inc.

DNA sequence analysis ofhprt mutants persisting in peripheral blood of cynomolgus monkeys more than two years after ENU treatment

We have been studying in vivo mutagenesis at the hypoxanthine phosphoribosyl transferase (hprt) locus in cynomolgus monkey T-lymphocytes. This primate model allows us to study mutations and their kinetics under well-controlled conditions. Previously, we reported mutations detected at various times after intraperitoneal treatment with ethylnitrosourea (ENU, 77 mg/kg). At 832 days after that first treatment, the monkey received a second dose of 77 mg/kg ENU. Up to 1,331 days after the second treatment, the T-cell mutant frequency (44.2 x 10(-6)) was still 26-fold higher than background (1.7 x 10(-6)), suggesting that mutants persisted in the peripheral blood. Mutant clones from Days 974, 1,164, and 1,311 after the second treatment were selected in thioguanine. Hprt cDNA was prepared from a cell lysate, PCR-amplified, and sequenced. Of 45 mutants, 30 yielded PCR product and 26 were sequenced. Base substitutions were found in 21 (81%) of the 26 mutants and consisted of one G:C --> A:T and five A:T --> G:C transitions, one G:C --> C:G, eight A:T --> T:A, and six A:T --> C:G transversions. Therefore, most base substitutions occurred at A:T basepairs, characteristic of ENU-induced mutations in vivo, and were detected up to 3.6 years after the second treatment. Deletions of exons 2 and 3 occurred in two mutants and exon 7 was deleted in one mutant. There were two insertion mutants: one was a single base insertion and the other contained an insertion of 277 basepairs which was nearly identical to a simian retroviral sequence.

DNA Mutation Detection Using Denaturing High‐Performance Liquid Chromatography (DHPLC)

AbstractThis method permits the rapid detection of singlebase substitutions and insertions or deletions of up to 1.5 kb in PCRamplified DNA fragments. The method exploits the differential retention of homo and heteroduplex DNA species under conditions of partial thermal denaturation and provides an efficient method to rapidly compare DNA fragments for sequence variation, once this variation has been identified by conventional sequencing.

Mutations in the genomic deoxyribonucleic acid for SLC3A1 in patients with cystinuria.

Cystinuria is an inherited transport disorder characterized by defective renal resorption of cystine and other dibasic amino acids. We have studied the occurrence of mutations in the SLC3A1 gene, which codes for a dibasic amino acid transporter-like protein, in 33 unrelated cystinurics. We found mutations in 34 of the 66 chromosomes studied. There were 14 different mutations in our study population, 8 of which had not been previously described. Of these new mutations, 4 were missense mutations: G1934C, C1259G, T1607G, and G1373A. The other 4 mutations consisted of a single base insertion mutation (2022 ins T), a single base deletion mutation (163 del C), a 23-base deletion mutation (del 782A-804A), and a complex mutation that consisted of a 36-base deletion (del C431-3 to T463) and a duplication insertion of 468 T to 474 A after nucleotide 474.

A novel splice site mutation in the tissue inhibitor of the metalloproteinases-3 gene in Sorsby's fundus dystrophy with unusual clinical features.

Sorsby's fundus dystrophy (SFD) is an autosomal dominant macular dystrophy which is developed usually in the third or fourth decade of life, and is characterized by central visual loss and nyctalopia due to fundus changes of exudative or atrophic macular lesions. Its functional prognosis is usually poor because of disciform macular scars and peripheral chorioretinal atrophies. To date, five different mutations in the tissue inhibitor of the metalloproteinases-3 (TIMP3) gene have been identified in families of a wide geographic origin, all of which are missense mutations that cause replacement by cysteine of conserved amino acids in the C-terminus of exon 5 of TIMP3. We have studied two Japanese families with SFD, the first report from the Eastern world, and identified a novel 3' splice site mutation in the TIMP3 gene, namely a single base insertion at the intron 4/exon 5 junction which converts the consensus sequence CAG to CAAG in the splice acceptor site. In addition, our patients displayed a distinctive clinical expression in that they developed macular dystrophies at an approximately 30-year later age of onset and preserved functional vision until later life with essentially uninvolved peripheral retina. The present findings may provide some insight into the genotype-phenotype relationship in SFD.

Mutation analysis in 57 unrelated patients with MPS II (Hunter’s disease)

Genomic DNA from 57 unrelated MPS II (Hunter’s disease) patients was analysed for mutations of the iduronate sulphatase (IDS) gene. The aim of the study was threefold: to identify the...

A novel insertion mutation (1286insC) in exon 9 of the factor XIII-A subunit gene.

Molecular studies have been performed on a Greek family with factor XIII-A subunit deficiency. The 15 exons of the A subunit gene were amplified by polymerase chain reaction and analysed by direct nucleotide sequencing. A homozygous single base insertion (1286insC) in exon 9 of the gene was identified in three affected family members. The insertion results in a frameshift and a premature stop signal a short distance downstream at codon 403. Any A subunit protein expressed is likely to be unstable and lack part of the catalytic core domain together with both beta barrel domains towards the C-terminal of the molecule. This study contributes to our knowledge of the mutational spectrum in patients with factor XIII-A deficiency.

Molecular Cloning and Expression Analysis of Five Novel Genes in Chromosome 1p36

The human chromosome 1p36 region displays frequent nonrandom chromosomal deletions and translocations in a number of human malignancies; these are thought to inactivate tumor suppressor genes. To identify these putative tumor suppressors we employed exon trapping, cDNA selection, and zoo blot analysis to clone five new genes located in 1p36. Two of these represent novel genes and were designated C1orf1 and xylan 1,4-β-xylosidase 1 (XBX1). Two further genes represented new members of known gene families: PTPRZ2 was a tyrosine phosphatase and FRAP2 represented a FKBP12–rapamycin-associated protein. The fifth gene identified, ENO1L1, was significantly homologous to c-myc promoter binding protein, MBP-1, and to enolase 1 (ENO1). It colocalized with α enolase (ENO1) on a single P1 clone. ENO1L1 differed from both ENO1 and MBP-1 in the organization of its 5′ untranslated sequences. Second, MBP-1 contained two single-base insertions not present in either ENO1 or ENO1L1 sequences, which led to a shift in the MBP-1 reading frame. Expression analysis revealed two brain-specific transcripts of 7.9 and 6.5 kb for PTPRZ2. In contrast, C1orf1, FRAP2, ENO1L1, and XBX1 appeared to be expressed ubiquitously in the tissues tested, with transcript sizes of 4.5, 8.7, 1.75, and 4.5 kb, respectively. Using fluorescencein situhybridization, we mapped the five novel genes relative to chromosome 1p36 breakpoints present in three established tumor cell lines and one nontumor cell line. The karyotypic abnormalities in these cell lines were exploited as chromosomal landmarks; we could thus show that the telomere to centromere gene order was PTPRZ2–(MBP-1/ENO1/ENO1L1)–(C1orf1/XBX1)–FRAP2. The localization of these genes to a chromosomal region that is prone to deletions in human cancers makes them potential candidate tumor suppressors.

An array of novel murine spleen focus-forming viruses that activate the erythropoietin receptor.

The Friend spleen focus-forming virus (SFFV) env gene encodes a 409-amino-acid glycoprotein with an apparent Mr of 55,000 (gp55) that binds to erythropoietin receptors (EpoR) to stimulate erythroblastosis. We reported previously the in vivo selection during serial passages in mice of several evolutionary intermediates that culminated in the formation of a novel SFFV (M. E. Hoatlin, E. Gomez-Lucia, F. Lilly, J. H. Beckstead, and D. Kabat, J. Virol. 72:3602-3609, 1998). A mouse injected with a retroviral vector in the presence of a nonpathogenic helper virus developed long-latency erythroblastosis, and subsequent viral passages resulted in more pathogenic isolates. The viruses taken from these mice converted an erythropoietin-dependent cell line (BaF3/EpoR) into factor-independent derivatives. Western blot analysis of cell extracts with an antiserum that broadly reacts with murine retroviral envelope glycoproteins suggested that the spleen from the initial mouse with mild erythoblastosis contained an array of viral components that were capable of activating EpoR. DNA sequence analysis of the viral genomes cloned from different factor-independent cell clones revealed env genes with open reading frames encoding 644, 449, and 187 amino acids. All three env genes contained 3' regions identical to that of SFFV, including a 6-bp duplication and a single-base insertion that have been shown previously to be critical for pathogenesis. However, the three env gene sequences did not contain any polytropic sequences and were divergent in their 5' regions, suggesting that they had originated by recombination and partial deletions of endogenously inherited MuLV env sequences. These results suggest that the requirements for EpoR activation by SFFV-related viruses are dependent on sequences at the 3' end of the env gene and not on the polytropic regions or on the 585-base deletions that are common among the classical strains of SFFV. Moreover, sequence analysis of the different recombinants and deletion mutants revealed that short direct and indirect repeat sequences frequently flanked the deletions that had occurred, suggesting a reverse transcriptase template jumping mechanism for this rapid retroviral diversification.