A 1.5-Mb deletion in 17p11.2-p12 is frequently observed in Italian families with hereditary neuropathy with liability to pressure palsies.

Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant disorder characterized by recurrent mononeuropathies. A 1.5-Mb deletion in chromosome 17p11.2-p12 has been associated with HNPP. Duplication of the same 1.5-Mb region is known to be associated with Charcot-Marie-Tooth disease type 1 (CMT1A), a more severe peripheral neuropathy characterized by symmetrically slowed nerve conduction velocity (NCV). The CMT1A duplication and HNPP deletion appear to be the reciprocal products of a recombination event involving a repeat element (CMT1A-REP) that flanks the 1.5-Mb region involved in the duplication/deletion. Patients from nine unrelated Italian families who were diagnosed with HNPP on the basis of clinical, electrophysiological, and histological evaluations were analyzed by molecular methods for DNA deletion on chromosome 17p. In all nine families, Southern analysis using a CMT1A-REP probe detected a reduced hybridization signal of a 6.0-kb EcoRI fragment mapping within the distal CMT1A-REP, indicating deletion of one copy of CMT1A-REP in these HNPP patients. Families were also typed with a polymorphic (CA)n repeat and with RFLPs corresponding to loci D17S122, D17S125, and D17S61, which all map within the deleted region. Lack of allelic transmission from affected parent to affected offspring was observed in four informative families, providing an independent indication for deletion. Furthermore, pulsed-field gel electrophoresis analysis of SacII-digested genomic DNA detected junction fragments specific to the 1.5-Mb HNPP deletion in seven of nine Italian families included in this study. These findings suggest that a 1.5-Mb deletion on 17p11.2-p12 is the most common mutation associated with HNPP.     

Molecular genetic analysis of the 17p11.2 region in patients with hereditary neuropathy with liability to pressure palsies (HNPP)

Hereditary neuropathy with liability to pressure palsies (HNPP) is in most cases associated with an interstitial deletion of the same 1.5-Mb region at 17p11.2 that is duplicated in Charcot-Marie-Tooth type 1A (CMT1A) patients. Unequal crossing-over following misalignment at flanking repeat sequences (CMT1A-REP), either leads to tandem duplication in CMT1A patients or deletion in HNPP patients. With the use of polymorphic DNA markers located within the CMT1A/HNPP duplication/deletion region we detected the HNPP deletion in 16 unrelated HNPP patients, 11 of Belgian and 5 of French origin. In all cases, the 1.5-Mb size of the HNPP deletion was confirmed by EcoRI dosage analysis using a CMT1A-REP probe. In the 16 HNPP patients, the same 370/320-kb EagI deletion-junction fragments were detected with pulsed field gel electrophoresis (PFGE), while in CMT1A patients, a 150-kb EagI duplication-junction fragment was seen. Thus, PFGE analysis of EagI-digested DNA with a CMT1A-REP probe allows direct detection of the HNPP deletion or the CMT1A duplication for DNA diagnostic purposes.     

Human meiotic recombination products revealed by sequencing a hotspot for homologous strand exchange in multiple HNPP deletion patients.

The HNPP (hereditary neuropathy with liability to pressure palsies) deletion and CMT1A (Charcot-Marie-Tooth disease type 1A) duplication are the reciprocal products of homologous recombination events between misaligned flanking CMT1A-REP repeats on chromosome 17p11. 2-p12. A 1.7-kb hotspot for homologous recombination was previously identified wherein the relative risk of an exchange event is 50 times higher than in the surrounding 98.7% identical sequence shared by the CMT1A-REPs. To refine the region of exchange further, we designed a PCR strategy to amplify the recombinant CMT1A-REP from HNPP patients as well as the proximal and distal CMT1A-REPs from control individuals. By comparing the sequences across recombinant CMT1A-REPs to that of the proximal and distal CMT1A-REPs, the exchange was mapped to a 557-bp region within the previously identified 1.7-kb hotspot in 21 of 23 unrelated HNPP deletion patients. Two patients had recombined sequences suggesting an exchange event closer to the mariner-like element previously identified near the hotspot. Five individuals also had interspersed patches of proximal or distal repeat specific DNA sequence indicating potential gene conversion during the exchange of genetic material. Our studies provide a direct observation of human meiotic recombination products. These results are consistent with the hypothesis that minimum efficient processing segments, which have been characterized in Escherichia coli, yeast, and cultured mammalian cells, may be required for efficient homologous meiotic recombination in humans.     

Polymorphisms in the PMP-22 gene region (17p11.2–12) are crucial for simplified diagnosis of duplications/deletions

DNA duplications and deletions of a 1.5-Mb region in chromosome 17p11.2–12 comprising the gene encoding peripheral myelin protein 22 (PMP-22) are the common mutations in Charcot-Marie-Tooth disease type 1 (CMT1) and hereditary neuropathy with liability to pressure palsies (HNPP). A 1.7-kb recombination hotspot region has been identified within misaligned flanking repeats (CMT1-REP elements) by detection of CMT- and HNPP-specific junction fragments in Southern blot analyses. In order to simplify routine diagnosis we introduce a polymerase chain reaction-based method to identify directly specific REP junction fragments. Using this test, specific fragments were detected in ∼ 67% of both CMT duplication and HNPP deletion cases. Polymorphism within a specific restriction enzyme recognition site is crucial for both Southern blot and PCR analyses of junction fragments. Received: 25 October 1996 / Revised: 16 December 1996     

Molecular evolution of the CMT1A-REP region: a human- and chimpanzee-specific repeat.

The CMT1A-REP repeat consists of two copies of a 24-kb sequence on human chromosome 17p11.2-12 that flank a 1.5-Mb region containing a dosage-sensitive gene, peripheral nerve protein-22 (PMP22). Unequal meiotic crossover mediated by misalignment of proximal and distal copies of the CMT1A-REP in humans leads to a 1.5-Mb duplication or deletion associated with two common peripheral nerve diseases, Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP). Previous molecular hybridization studies with CMT1A-REP sequences suggested that two copies of the repeat are also found in the chimpanzee, raising the possibility that this unique repeat arose during primate evolution. To further characterize the structure and evolutionary synthesis of the CMT1A-REP repeat, fluorescent in situ hybridization (FISH) analysis and heterologous PCR-based assays were carried out for a series of primates. Genomic DNA was analyzed with primers selected to differentially amplify the centromeric and telomeric ends of the human proximal and distal CMT1A-REP elements and an associated mariner (MLE) sequence. All primate species examined (common chimpanzee, pygmy chimpanzee, gorilla, orangutan, gibbon, baboon, rhesus monkey, green monkey, owl monkey, and galago) tested positive for a copy of the distal element. In addition to humans, only the chimpanzee was found to have a copy of the proximal CMT1A-REP element. All but one primate species (galago) tested positive for the MLE located within the CMT1A-REP sequence. These observations confirm the hypothesis that the distal CMT1A-REP element is the ancestral sequence which was duplicated during primate evolution, provide support for a human-chimpanzee clade, and suggest that insertion of the MLE into the CMT1A-REP sequence occurred in the ancestor of anthropoid primates.     

Mosaicism for the Charcot-Marie-Tooth disease type 1A duplication suggests somatic reversion

A female patient with clinical signs and symptoms of a demyelinating neuropathy was shown to have a duplication of the 1.5-Mb region on chromosome 17p11.2, typical of the great majority of cases of Charcot-Marie-Tooth disease type 1A (CMT1A). However, analysis of DNA extracted from peripheral blood revealed a 2:2.4 instead of the usual 2:3 ratio between the 7.8- and 6.0-kb EcoRI fragments in the proximal and distal repetitive extragenic palindromic (REP) elements of CMT1A. Detection of a 3.2-kb EcoRI/SacI kb junction fragment with probe pLR7.8 confirmed the CMT1A duplication. The dosage of this junction fragment, compared with a 2.8-kb EcoRI/SacI fragment of the proximal REP elements of CMT1A, was 2:0.58 instead of the expected 2:1 dosage for heterozygous CMT1A duplications. We hypothesized that the lower dosages of these restriction fragments specific for the CMT1A duplication were due to mosaicism; this was confirmed by fluorescence in situ hybridization analysis with the D17S122-specific probe pVAW409R1. In peripheral blood lymphocytes the percentage of interphase nuclei with a duplication in 17p11.2 was 49%. In interphase nuclei extracted from buccal mucosa, hair-root cells or paraffin-embedded nervous tissue the duplication was detectable in 51%, 66% and 74%, respectively. This is the first report of mosaicism in a patient with a CMT1A duplication identified by three different and independent techniques. Received: 14 November 1995 / Revised: 13 February 1996     

Rapid diagnosis of CMT1A duplications and HNPP deletions by multiplex microsatellite PCR

Charcot-Marie-Tooth (CMT) disease and hereditary neuropathy with liability to pressure palsies (HNPP) are frequent forms of genetically heterogeneous peripheral neuropathies. Reciprocal unequal crossover between flanking CMT1A-REPs on chromosome 17p11.2-p12 is a major cause of CMT type 1A (CMT1A) and HNPP. The importance of a sensitive and rapid method for identifying the CMT1A duplication and HNPP deletion is being emphasized. In the present study, we established a molecular diagnostic method for the CMT1A duplication and HNPP deletion based on hexaplex PCR of 6 microsatellite markers (D17S921, D17S9B, D17S9A, D17S918, D17S4A and D17S2230). The method is highly time-, cost- and sample-saving because the six markers are amplified by a single PCR reaction and resolved with a single capillary in 3 h. Several statistical and forensic estimates indicated that most of these markers are likely to be useful for diagnosing the peripheral neuropathies. Reproducibility, as determined by concordance between independent tests, was estimated to be 100%. The likelihood that genotypes of all six markers are homozygous in randomly selected individuals was calculated to be 1.6 x 10(-4) which indicates that the statistical error rate for this diagnosis of HNPP deletion is only 0.016%.     

Mutational analysis of the MPZ, PMP22 and Cx32 genes in patients of Spanish ancestry with Charcot-Marie-Tooth disease and hereditary neuropathy with liability to pressure palsies

Charcot-Marie-Tooth disease (CMT) and hereditary neuropathy with liability to pressure palsies (HNPP) are two inherited peripheral neuropathies. The most prevalent mutations are a reciprocal 1.5-Mb duplication and 1.5-Mb deletion, respectively, at the CMT1A/HNPP locus on chromosome 17p11.2. Point mutations in the coding region of the myelin genes, peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ) or connexin 32 (Cx32) have been reported in CMT patients, including CMT type 1 (CMT1), CMT type 2 (CMT2) and Déjérine-Sottas neuropathy (DS) patients, and only in the coding region of PMP22 in HNPP families lacking a deletion. We have investigated point and small mutations in the MPZ, PMP22 and Cx32 genes in a series of patients of Spanish ancestry: 47 CMT patients without duplications, and 5 HNPP patients without deletions. We found 15 different mutations in 16 CMT patients (34%). Nine different mutations in ten patients were detected in the Cx32 gene, this being the most frequently involved gene in this series, whereas five mutations involved the MPZ gene and only one the PMP22 gene. Six out of nine nucleotide substitutions in the Cx32 gene involved two codons encoding arginine at positions 164 and 183, suggesting that these two codons may constitute two Cx32 regions prone to mutate in the Spanish population. Analysis of HNPP patients revealed a 5′ splicing mutation in intron 1 of the PMP22 gene in a family with autosomal dominance, which confirms allelic heterogeneity in HNPP. Ectopic mRNA analysis on leukocytes suggests that this mutation might behave as a null allele. Received: 25 July 1996 / Revised: 15 November 1996     

Polymorphic class II sequences linked to the rat major histocompatibility complex (RT1) homologous to human DR and DQ sequences

Until recently, the analysis of Class II genes linked to the rat major histocompatibility complex, RT1, has been confined to serologic and electrophoretic analysis of their gene products. To obtain a more definitive estimate of the number and relative polymorphism of RT1 Class II sequences, we performed Southern blot analysis of rat genomic DNA employing human cDNA probes specific for Class II heavy and light chain genes. Southern blots of EcoRI and BamHI digests of genomic DNA from ten inbred strains, expressing eight RT1 haplotypes, were hybridized with the human DQ beta or DR beta cDNA that are homologous to Class II light chain sequences. Four to eight bands were observed to hybridize with the light chain cDNA: band sizes ranged from 2.5 to 28 kb. Restriction fragment patterns were polymorphic; the only identical patterns observed were those associated with RT1 haplotypes with identical RT1.B regions. The number and size of bands hybridizing with DQ beta and DR beta suggested a minimum of four light chain sequences in each haplotype. Southern blots of BamHI and EcoRI digests of genomic DNA from the same strains were hybridized with a DR alpha cDNA that is homologous to Class II heavy chain sequences. All RT1 haplotypes expressed either a 10.0-kb or 13.0-kb band when digested with BamHI, and either a 17-kb or 3.7-kb band when digested with EcoRI. Considerably less polymorphism was detected with the DR alpha probe; this observation is consistent with previously reported limited protein polymorphism of the rat equivalent of the I-E alpha subunit. The size and number of bands hybridizing with the DR alpha probe suggests a minimum of two heavy chain sequences. These observations suggest that the RT1 complex includes more Class II sequences than have been observed in serologic and electrophoretic analyses of Class II gene products. Furthermore, the level of polymorphism of RT1 Class II sequences appears to be comparable with mouse and human Class II sequences.     

Localization of Charcot-Marie-Tooth disease type 1a (CMT1A) to chromosome 17p11.2.

Charcot-Marie-Tooth (CMT) disease type 1a has been previously localized to chromosome 17 using the markers D17S58 and D17S71. In that report we were unable to provide unequivocal localization of the CMT1A gene on either the proximal p or the q arm. Therefore, data from one additional CMT1A family and typing of other probes spanning the pericentromeric region of chromosome 17 (D17S73, D17S58, D17S122, D17S125, D17S124) were analyzed. Multipoint analysis demonstrates convincing evidence (log likelihood difference greater than 5) that the CMT1A gene lies within 17p11.2 and most likely between the flanking markers D17S122 and D17S124.     

Gene conversion homogenizes the CMT1A paralogous repeats

Background

Non-allelic homologous recombination between paralogous repeats is increasingly being recognized as a major mechanism causing both pathogenic microdeletions and duplications, and structural polymorphism in the human genome. It has recently been shown empirically that gene conversion can homogenize such repeats, resulting in longer stretches of absolute identity that may increase the rate of non-allelic homologous recombination.

Results

Here, a statistical test to detect gene conversion between pairs of non-coding sequences is presented. It is shown that the 24 kb Charcot-Marie-Tooth type 1A paralogous repeats (CMT1A-REPs) exhibit the imprint of gene conversion processes whilst control orthologous sequences do not. In addition, Monte Carlo simulations of the evolutionary divergence of the CMT1A-REPs, incorporating two alternative models for gene conversion, generate repeats that are statistically indistinguishable from the observed repeats. Bounds are placed on the rate of these conversion processes, with central values of 1.3 × 10^-4 and 5.1 × 10^-5 per generation for the alternative models.

Conclusions

This evidence presented here suggests that gene conversion may have played an important role in the evolution of the CMT1A-REP paralogous repeats. The rates of these processes are such that it is probable that homogenized CMT1A-REPs are polymorphic within modern populations. Gene conversion processes are similarly likely to play an important role in the evolution of other segmental duplications and may influence the rate of non-allelic homologous recombination between them.

     

242 breakpoints in the 200-kb deletion-prone P20 region of the DMD gene are widely spread.

Using whole cosmids as probes, we have mapped 242 DMD/BMD deletion breakpoints located in the major deletion hot spot of the DMD gene. Of these, 113 breakpoints were mapped more precisely to individual restriction enzyme fragments in the distal 80 kb of the 170-kb intron 44. An additional 12 breakpoints are distributed over the entire region, with no significant local variation in frequency. Furthermore, deletion sizes vary and are not influenced by the positions of the breakpoints. This argues against a predominant role of one or a few specific sequences in causing frequent rearrangements. It suggests that structural characteristics or a more widespread recombinogenic sequence makes this region so susceptible to deletion. Our study revealed several RFLPs, one of which is a 300-bp insertion/deletion polymorphism. Abnormally migrating junction fragments are found in 81% of the precisely mapped deletions and are highly valuable in the diagnosis of carrier females.     

Simultaneously arising Ly-1(CD5) B cell lymphomas have identical expressed IgH and kappa-genes but different nonproductive heavy chain rearrangements.

A group of CD5(Ly-1) B cell lymphomas are described. They were derived from mice which received a common pool of syngeneic mouse spleen cells. Southern blot analysis revealed that the lymphomas exhibited an unusual set of Ig gene rearrangements. Six lymphomas analyzed had either of two rearrangement patterns. EcoRI restriction digests of tumor DNA probed for rearrangements in the JH region, resulted in restriction fragments of 4.7 and 5.6 kb or of 4.7 and 8.5 kb. Each had an identical HindIII restriction fragment identified when probed for kappa gene rearrangements. Inasmuch as several B cell lymphomas from mice receiving a common pool of spleen cells had identical kappa-rearrangements and one identical IgH rearrangement, it was important to determine the DNA sequence of expressed IgH and kappa-genes. Each tumor was found to have identical nucleotide sequences of VH-DH-JH and VK-JK. The nonproductive IgH rearrangements each consisted of incomplete DH-JH rearrangements. The 8.5-kb EcoRI fragment was generated from a DFL16 gene segment rearranged into JH3, and the 5.6-kb fragment was generated from DQ52 rearranged into JH)1. We conclude that these Ly-1 B tumors are most likely derived from a single clone of cells which underwent a secondary rearrangement on the nonproductive allele after kappa-rearrangement had occurred. The alternate possibility of independently arising lymphomas with identical expressed VH and VK sequences is discussed.     

A unique point mutation in the PMP22 gene is associated with Charcot-Marie-Tooth disease and deafness.

Charcot-Marie-Tooth disease (CMT) with deafness is clinically distinct among the genetically heterogeneous group of CMT disorders. Molecular studies in a large family with autosomal dominant CMT and deafness have not been reported. The present molecular study involves a family with progressive features of CMT and deafness, originally reported by Kousseff et al. Genetic analysis of 70 individuals (31 affected, 28 unaffected, and 11 spouses) revealed linkage to markers on chromosome 17p11.2-p12, with a maximum LOD score of 9.01 for marker D17S1357 at a recombination fraction of .03. Haplotype analysis placed the CMT-deafness locus between markers D17S839 and D17S122, a approximately 0.6-Mb interval. This critical region lies within the CMT type 1A duplication region and excludes MYO15, a gene coding an unconventional myosin that causes a form of autosomal recessive deafness called DFNB3. Affected individuals from this family do not have the common 1.5-Mb duplication of CMT type 1A. Direct sequencing of the candidate peripheral myelin protein 22 (PMP22) gene detected a unique G-->C transversion in the heterozygous state in all affected individuals, at position 248 in coding exon 3, predicted to result in an Ala67Pro substitution in the second transmembrane domain of PMP22.     

Multiple replicons constituting the genome of Pseudomonas cepacia 17616.

Macrorestriction fragment analysis of DNA from Pseudomonas cepacia 17616, in conjunction with Southern hybridization experiments using junction fragments containing rare restriction enzyme sites as probes, indicated that this bacterium contains three large circular replicons of 3.4, 2.5, and 0.9 megabases (Mb). Inclusion of the 170-kb cryptic plasmid present in this strain gave an overall estimate of genome size of 7 Mb. Other Southern hybridization experiments indicated that the three large replicons contained rRNA genes as well as insertion sequence elements identified previously in this strain. The distribution of SwaI, PacI, and PmeI sites on the three replicons was determined. A derivative of Tn5-751 carrying a SwaI site was used to inactivate and map genes on the 2.5- and 3.4-Mb replicons. Mutants were isolated in which the 2.5- and 0.9-Mb replicons had been reduced in size to 1.8 and 0.65 Mb, respectively. The loss of DNA from the 2.5-Mb replicon was associated with lysine auxotrophy, beta-lactamase deficiency, and failure to utilize ribitol and trehalose as carbon and energy sources. DNA fragments corresponding in size to randomly linearized forms of the different replicons were detected in unrestricted DNA by pulsed-field gel electrophoresis. The results provide a framework for further genetic analysis of strain 17616 and for evaluation of the genomic complexities of other P. cepacia isolates.     

DNA duplication associated with Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-tooth disease type 1A (CMT1A) was localized by genetic mapping to a 3 cM interval on human chromosome 17p. DNA markers within this interval revealed a duplication that is completely linked and associated with CMT1A. The duplication was demonstrated in affected individuals by the presence of three alleles at a highly polymorphic locus, by dosage differences at RFLP alleles, and by two-color fluorescence in situ hybridization. Pulsed-field gel electrophoresis of genomic DNA from patients of different ethnic origins showed a novel SacII fragment of 500 kb associated with CMT1A. A severely affected CMT1A offspring from a mating between two affected individuals was demonstrated to have this duplication present on each chromosome 17. We have demonstrated that failure to recognize the molecular duplication can lead to misinterpretation of marker genotypes for affected individuals, identification of false recombinants, and incorrect localization of the disease locus.     

Mechanisms for Nonrecurrent Genomic Rearrangements Associated with CMT1A or HNPP: Rare CNVs as a Cause for Missing Heritability

Genomic rearrangements involving the peripheral myelin protein gene (PMP22) in human chromosome 17p12 are associated with neuropathy: duplications cause Charcot-Marie-Tooth disease type 1A (CMT1A), whereas deletions lead to hereditary neuropathy with liability to pressure palsies (HNPP). Our previous studies showed that >99% of these rearrangements are recurrent and mediated by nonallelic homologous recombination (NAHR). Rare copy number variations (CNVs) generated by nonrecurrent rearrangements also exist in 17p12, but their underlying mechanisms are not well understood. We investigated 21 subjects with rare CNVs associated with CMT1A or HNPP by oligonucleotide-based comparative genomic hybridization microarrays and breakpoint sequence analyses, and we identified 17 unique CNVs, including two genomic deletions, ten genomic duplications, two complex rearrangements, and three small exonic deletions. Each of these CNVs includes either the entire PMP22 gene, or exon(s) only, or ultraconserved potential regulatory sequences upstream of PMP22, further supporting the contention that PMP22 is the critical gene mediating the neuropathy phenotypes associated with 17p12 rearrangements. Breakpoint sequence analysis reveals that, different from the predominant NAHR mechanism in recurrent rearrangement, various molecular mechanisms, including nonhomologous end joining, Alu-Alu-mediated recombination, and replication-based mechanisms (e.g., FoSTeS and/or MMBIR), can generate nonrecurrent 17p12 rearrangements associated with neuropathy. We document a multitude of ways in which gene function can be altered by CNVs. Given the characteristics, including small size, structural complexity, and location outside of coding regions, of selected rare CNVs, their identification remains a challenge for genome analysis. Rare CNVs may potentially represent an important portion of “missing heritability” for human diseases.     

Analysis of 17p11.2 chromosome region rearrangements in CMT1 patients from Ukraine

Two intercomplementary methods of 17p11.2 duplication/deletion identification have been elaborated: STR allelic variants analysis and direct PMP22 gene dosage measuring by means of quantitative Real-Time PCR. It has been carried out detection and analysis of 17p11.2 chromosome region rearrangements in CMT1 patients from Ukraine. It has been registered the high level of de novo cases with 17p11.2-duplication. It has been shown the 17p11.2 chromosome region duplication/deletion association with CMT1A and HNPP clinical phenotypes which may be used in differential diagnosis of this type of CMT polyneuropathy. The article is published in the original.     

Identification and localization of reiterated sequences in the Choristoneura fumiferana MNPV genome.

The genome of Choristoneura fumiferana nuclear polyhedrosis virus (CfMNPV) contained reiterated sequences interdispersed in four locations. These regions, termed RS, were found in EcoRI fragments A, F, E and B. The sequences were identified by hybridization of the fragment EcoRI-A to a Southern blot of EcoRI-digested viral DNA. Further confirmation and more precise localization of the RS sequences was obtained by hybridization of nick-translated 32P-labeled EcoRI-E fragment to Southern blots of viral DNA digested with EcoRI, BamHI, XbaI and Bg/II. Hybridization of 32P-labeled EcoRI-E to HindIII blots of viral DNA revealed the presence of a 'ladder' consisting of eight fragments. The three fragments of the ladder with the lowest sizes represented the HindIII fragments, O, PQ and R. The other five fragments were submolar in amount, in that they could not be seen in ethidium bromide-stained gels and probably represented minor virus variants that arose after passage of virus in larvae. Each variant was distinguished from the others by an additional insertion of 210 bp into the EcoRI-B fragment of the genome.