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Two previous case reports have described an apparently new lethal syndrome consisting of pachygyria, joint contractures and facial abnormalities (Winter et al., 1989; Tsukahara et al., 1990). Another report describes a non-lethal case in which dysmorphic features were not noted (Massa et al., 1988). We now report on what appears to be an additional lethal case. This male infant had a lethal condition with features of large fontanelle, pachygyria with incomplete opercularization, varus contractures of the hands and feet, small palpebral fissures, hypertelorism, a small penis, cryptorchidism, camptodactyly and a sandal gap deformity. These cases appear to represent a new lethal lissencephaly syndrome associated with arthrogryposis and facial dysmorphism, which we propose to call the Winter-Tsukahara syndrome.

Charcot-Marie-Tooth disease type 1A (CMT1A) is an autosomal dominant neuropathy that can be caused by dominant point mutations in PMP22 which encodes a peripheral nerve myelin protein. Usually, CMT1A is caused by the duplication of a 1.5-megabase (Mb) region on chromosome 17p11.2-p12 containing PMP22. Deletion of a similar 1.5-Mb region is associated with hereditary neuropathy with liability to pressure palsies (HNPP), a clinically distinct neuropathy. We have identified a severely affected CMT1 patient who is a compound heterozygote for a recessive PMP22 point mutation, and a 1.5 Mb deletion in 17p11.2-p12. A son heterozygous for the PMP22 point mutation had no signs of neuropathy, while two others heterozygous for the deletion had HNPP, suggesting that point mutations in PMP22 can result in dominant and recessive alleles contributing to CMT1A.

We describe an infant with del(17) (p11.2p12) whose deleted chromosome was inherited from a mosaic mother. The child had manifestations consistent with Smith-Magenis syndrome. The mother appeared to be of normal intelligence and she had minimal findings of Smith-Magenis syndrome. Separation of chromosome 17 homologues in somatic cell hybrids and molecular studies confirmed the cytogenetic diagnoses and the fact that the mother was mosaic. Furthermore, molecular analysis demonstrated novel breakpoints in this family, with the deletion extending into and completely encompassing the markers duplicated in Charcot-Marie-Tooth (CMT) disease. Although this Smith-Magenis syndrome patient is completely deleted for the CMT region, her electrophysiological findings are different from those found in CMT. This is the only reported case of Smith-Magenis syndrome with transmission from a partially affected mosaic mother. Transmission of interstitial deletions from mosaic parents may be more common than thought; therefore, parental chromosomes should be examined when interstitial deletions are identified.

Charcot-Marie-Tooth disease (CMT) comprises a clinically and genetically heterogeneous group of polyneuropathies. Two major types can be distinguished based on electrophysiologic phenotypes: CMT type 1 (CMT1) displays uniformly decreased nerve conduction velocity associated with a demyelinating hypertrophic neuropathy, and CMT type 2 (CMT2) displays normal or near-normal nerve conduction velocity associated with a neuronal defect. Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common form, exhibiting autosomal dominant inheritance and linkage to chromosome 17p11.2p12. This review will focus on the underlying molecular mechanisms leading to CMT1A. DNA duplication of a 1.5-Mb region is associated with CMT1A in the majority of cases. A defined segmental DNA duplication that cosegregates with a disease in a dominant Mendelian pattern had been unprecedented. A candidate gene for CMT1A, PMP22, which maps within the duplication and encodes a myelin-specific protein, was identified from studies on the trembler and tremblerJ mouse models for CMT. Point mutations in PMP22 have since been identified in cases of familial, non-duplication CMT1A. The genetic data presents two alternative molecular mechanisms involving the PMP22 gene that result in the same clinical and electrophysiologic phenotype of CMT1A. The impact of the underlying molecular mechanisms on the prospects for therapeutic development are discussed.

We evaluated motor conduction velocities in a large group of patients and their unaffected kin from five families in which a segmental duplication of chromosome 17p has shown complete linkage to Charcot-Marie-Tooth disease type 1 (CMT1A). Slowing of conduction was completely concordant with the presence of the segmental duplication; two clinically normal patients had slowed conduction. Nonetheless, among the patients with the CMT1A duplication, conduction velocities varied widely, by > 30 m/sec overall, by > 20 m/sec within families, and often by more than 10 m/sec between siblings and between parents and children. One patient was homozygous for the chromosome 17p duplication and had the slowest conduction velocity observed. Conduction slowing was not age-dependent and was present early in childhood. Our findings demonstrate complete penetrance at an early age of the electrophysiologic phenotype associated with the chromosome 17p duplication and confirm the reliability of nerve conduction studies in establishing the affection status in CMT1A. The great variation in conduction velocity among CMT1A patients emphasizes the influence of factors apart from the shared genetic mutation on phenotypic expression.

Background: Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. CMT type 1A is associated with a 1.5-megabase DNA duplication in region p11.2-p12 of chromosome 17 in most patients. An increased dosage of a gene within the duplicated segment appears to cause the disease. The PMP22 gene, which encodes a myelin protein, has been mapped within the duplication and proposed as a candidate gene for CMT type 1A., Methods: We analyzed DNA samples from a cohort of 32 unrelated patients with CMT type 1 who did not have the 1.5-Mb tandem duplication in 17p11.2-p12 for mutations within the PMP22 coding region. Molecular techniques included the polymerase chain reaction (PCR), heteroduplex analysis to detect point mutations, and direct nucleotide-sequence determination of amplified PCR products., Results: A 10-year-old boy was identified with a point mutation in PMP22, which resulted in the substitution of cysteine for serine in a putative transmembrane domain of PMP22. Analysis of family members revealed that the PMP22 point mutation arose spontaneously and segregated with the CMT type 1 phenotype in an autosomal dominant pattern. The patients with the PMP22 point mutation had clinical and electrophysiologic phenotypes that were similar to those of patients with the 1.5-Mb duplication., Conclusions: The PMP22 gene has a causative role in CMT type 1. Either a point mutation in PMP22 or a duplication of the region including the PMP22 gene can result in the disease phenotype.

Repetitive element sequence-based polymerase chain reaction (rep-PCR) enables the generation of DNA fingerprints which discriminate bacterial species and strains. We describe the application of whole-cell methods which allow specimens from broth cultures or colonies from agar plates to be utilized directly in the PCR mixture. The rep-PCR-generated DNA fingerprints obtained with whole-cell samples match results obtained with genomic DNA templates. Examples with different gram-negative bacteria (e.g., Citrobacter diversus, Escherichia coli, and Pseudomonas aeruginosa) and gram-positive bacteria (e.g., Staphylococcus aureus and Streptococcus pneumoniae) are demonstrated. Rapid specimen preparation methods enable rep-PCR-based fingerprinting to be completed in several hours and, therefore, allows the timely analysis of epidemiological relationships.

The macromolecular synthesis (MMS) operon contains three essential genes (rpsU, dnaG, rpoD) whose products (S21, primase, sigma-70) are necessary for the initiation of protein, DNA, and RNA synthesis respectively. PCR amplifications with primers complementary to conserved regions within these three genes, and subsequent DNA sequencing of rpsU-dnaG PCR products, demonstrate that the three genes appear to be contiguous in 11 different Gram-negative species. Within the Gram-negative enteric bacterial lineage, the S21 amino acid sequence is absolutely conserved in 10 species examined. The putative nuteq antiterminator sequence in rpsU consists of two motifs, boxA and boxB, conserved in primary sequence and secondary structure. The terminator sequence, T1, located between rpsU and dnaG is conserved at 31 positions in nine enterobacterial species, suggesting the importance of primary sequence in addition to secondary structure for transcription termination. The intergenic region between rpsU and dnaG varies in size owing to the presence or absence of the Enterobacterial Repetitive Intergenic Consensus (ERIC) DNA element. The rpoD gene contains rearrangements involving a divergent sequence, although two carboxy-terminal regions which encode functional domains are conserved in primary sequence and spacing. Our data suggest that primary sequence divergence and DNA rearrangements in both coding and non-coding sequences account for the interspecies variation in operon structure. However, MMS operon gene organization and cis-acting regulatory sequences appear to be conserved in diverse bacteria.

A sample of 48 penicillin-resistant Streptococcus pneumoniae (PRSP) strains recovered between January 1989 and May 1991, primarily from infected children in Houston, was characterized by multilocus enzyme electrophoresis and repetitive extragenic palindromic-polymerase chain reaction genomic profiling. A heterogeneous array of 22 clonal genotypes was identified, but 64% of the PRSP strains in the sample were assigned to five clones that are closely similar in overall chromosomal character and express serotype 6 capsule. A close genetic association between these five clones and penicillin-resistant 6B clones recovered in Alaska, Iceland, and Spain was identified by multilocus enzyme electrophoresis. Taken together, the results suggest either that the common resistant 6B clones in Alaska, Iceland, Spain, and Houston have a recent ancestor or that isolates of a certain pneumococcal phylogenetic lineage are more likely to develop penicillin resistance.

Charcot-Marie-Tooth disease type 1A (CMT1A) was recently demonstrated to be associated with a large DNA duplication in 17p11.2p12. The gene for neurofibromatosis type 1 (NF1) or von Recklinghausen disease maps to 17q11.2. We have identified 2 unrelated patients who were diagnosed with both CMT1 and NF1. Molecular analysis of these patients demonstrated the presence of the CMT1A duplication and inheritance of this DNA rearrangement from a parent affected with CMT. Analysis of genomic DNA isolated from the neurofibroma removed from one of these patients showed the same 500 kb SacII junction fragment associated with the CMT1A duplication that was found in genomic DNA isolated from the blood. These results lend further support to the hypothesis that the CMT1A duplication is a stable DNA rearrangement. In addition, the molecular analysis of these 2 patients suggests that 2 common autosomal dominant conditions (CMT1 and NF1) can occur in the same individual, not because of an underlying single molecular defect, but rather, secondary to a chance phenomenon.

We have constructed a 3.1 megabase (Mb) physical map of chromosome 17p11.2-p12, which contains a submicroscopic duplication in patients with Charcot-Marie-Tooth disease type 1A (CMT1A). We find that the CMT1A duplication is a tandem repeat of 1.5 Mb of DNA. A YAC contig encompassing the CMT1A duplication and spanning the endpoints was also developed. Several low copy repeats in 17p11.2-p12 were identified including the large (> 17 kb) CMT1A-REP unit which may be part of a mosaic repeat. CMT1A-REP flanks the 1.5 Mb CMT1A monomer unit on normal chromosome 17 and is present in an additional copy on the CMT1A duplicated chromosome. We propose that the de novo CMT1A duplication arises from unequal crossing over due to misalignment at these CMT1A-REP repeat sequences during meiosis.

Oligonucleotide probes which match consensus sequences of the repetitive extragenic palindromic (REP) element hybridize to genomic DNA of diverse bacterial species. Primers based on the REP sequence generate complex band patterns with genomic DNA in the polymerase chain reaction (PCR), a technique named REP-PCR. We used REP-PCR with genomic DNA to fingerprint 47 isolates of Citrobacter diversus. Previously, 37 were assigned electrophoretic types (ETs) by multilocus enzyme electrophoresis and 35 were evaluated by using outer membrane protein profiles. Fingerprints were compared by visual inspection and by similarity coefficients (SimCs) based on the number of common bands versus total bands between two given isolates. DNA fingerprints were highly similar visually for patient pairs and outbreak-related sets. SimCs for these were > or = 0.952. Fingerprints of isolates with different ETs generally were distinctive. Among 21 unrelated isolates representing 15 ETs, only 6 of 210 comparisons had SimCs of > or = 0.952. REP-PCR rapidly generated DNA fingerprints which were highly similar for epidemiologically linked isolates of C. diversus and distinct for previously characterized strains within this species. The ability of this method to discriminate between C. diversus isolates with the same biotype was similar to that of multilocus enzyme electrophoresis and outer membrane protein profiles. REP-PCR may be useful in evaluation of apparent outbreaks of this or other bacterial species which possess these extragenic, repetitive elements.

Charcot-Marie-Tooth (CMT) syndrome describes a genetically and clinically heterogeneous group of polyneuropathies. Electrophysiologically, at least two types of CMT can be distinguished; CMT1 which has decreased nerve conduction velocities (NCV) and CMT2 which has normal or near normal NCV with decreased amplitudes. For CMT1, three gene loci (on chromosomes 1, 17 and the X chromosome) have been mapped. The locus on chromosome 17, CMT type 1A (CMT1A), is responsible for the most common form of CMT which has recently been shown to be associated with a large DNA duplication. Recent data demonstrates that the CMT1A phenotype results from an inherited DNA rearrangement and a gene dosage effect. The trembler (Tr) and allelic tremblerJ (TrJ) mice have been proposed as animal models for CMT. Tr has similar electrophysiological and neuropathological features to CMT1 patients and maps to mouse chromosome 11 in a region of conserved synteny with human chromosome 17p. Tr and TrJ have recently been shown to have different point mutations in regions encoding putative transmembrane domains of the myelin specific protein PMP-22. The human peripheral nerve-specific PMP-22 gene maps within the CMT1A duplication. PMP-22 is thus a candidate gene for CMT1A. This paper describes the molecular genetics of CMT1A and sural nerve pathology in CMT1A patients with the CMT1A duplication.

Somatic cell hybrids retaining the deleted chromosome 17 from 15 unrelated Smith-Magenis syndrome (SMS) [del(17)(p11.2p11.2)] patients were obtained by fusion of patient lymphoblasts with thymidine kinase-deficient rodent cell lines. Seventeen sequence-tagged sites (STSs) were developed from anonymous markers and cloned genes mapping to the short arm of chromosome 17. The STSs were used to determine the deletion status of these loci in these and four previously described human chromosome 17-retaining hybrids. Ten STSs were used to identify 28 yeast artificial chromosomes (YACs) from the St. Louis human genomic YAC library. Four of the 17 STSs identified simple repeat polymorphisms. The order and location of deletion breakpoints were confirmed and refined, and the regional assignment of several probes and cloned genes were determined. The cytogenetic band locations and relative order of six markers on 17p were established by fluorescence in situ hybridization mapping to metaphase chromosomes. The latter data confirmed and supplemented the somatic cell hybrid results. Most of the hybrids derived from [del(17)(p11.2p11.2)] patients demonstrated a similar pattern of deletion for the marker loci and were deleted for D17S446, D17S258, D17S29, D17S71, and D17S445. However, one of them demonstrated a unique pattern of deletion. This patient is deleted for several markers known to recognize a large DNA duplication associated with Charcot-Marie-Tooth (CMT) disease type 1A. These data suggest that the proximal junction of the CMT1A duplication is close to the distal breakpoint in [del(17)(p-11.2p11.2)] patients.

Charcot-Marie-Tooth disease type 1A (CMT1A) is an autosomal dominant peripheral neuropathy associated with a large DNA duplication on the short arm of human chromosome 17. The trembler (Tr) mouse serves as a model for CMT1A because of phenotypic similarities and because the Tr locus maps to mouse chromosome 11 in a region of conserved synteny with human chromosome 17. Recently, the peripheral myelin gene Pmp-22 was found to carry a point mutation in Tr mice. We have isolated cDNA and genomic clones for human PMP-22. The gene maps to human chromosome 17p11.2-17p12, is expressed at high levels in peripheral nervous tissue and is duplicated, but not disrupted, in CMT1A patients. Thus, we suggest that a gene dosage effect involving PMP-22 is at least partially responsible for the demyelinating neuropathy seen in CMT1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited peripheral neuropathy in humans, characterized electrophysiologically by decreased nerve conduction velocities (NCVs). CMT1A is associated with a large submicroscopic DNA duplication in proximal 17p. In this report we demonstrate that a patient with a cytogenetically visible duplication, dup(17)(p11.2p12), has decreased NCV. Molecular analysis demonstrated this patient was duplicated for all the DNA markers duplicated in CMT1A as well as markers both proximal and distal to the CMT1A duplication. These data support the hypothesis that the CMT1A phenotype can result from a gene dosage effect.

Rod monochromacy (complete congenital achromatopsia) is inherited as an autosomal recessive trait of unknown genetic location. The disorder is characterized by total absence of color discrimination because retinal cone photoreceptors do not develop; systemic features do not occur. A 20-year-old white female with rod monochromacy presented with short stature (less than 5th percentile), mild developmental delay, premature puberty, small hands and feet (length less than 5th percentile), minimal dysmorphism, and a reproductive history of three consecutive first-trimester miscarriages. Cytogenetic analysis showed 45,XX,rob(14;14) in all 30 cells examined. Southern analysis of DNA from the patient and her phenotypically normal mother and two brothers (her father is deceased) ascertained the parental origin of the 14;14 Robertsonian translocation. Analysis of RFLPs associated with nine VNTR probes and two dinucleotide repeat polymorphisms from chromosome 14 demonstrated that the patient had inherited two copies of a single allele, each of which was maternally derived. A fully informative RFLP analysis of three probes from chromosome 14 enabled reconstruction of the paternal haplotype and showed the lack of any paternal contribution to the subject. These data are consistent with maternal isodisomy for all portions of chromosome 14 tested by these markers. This finding suggests that rod monochromacy maps to chromosome 14, and it emphasizes the importance of uniparental isodisomy to provide a putative chromosomal assignment of a gene for a rare autosomal recessive disorder.

We describe a novel method to map chromosomal Escherichia coli::Tn5 insertion mutations rapidly. This method utilizes the ends of Tn5 and the E. coli REP sequence as primer binding sites for the polymerase chain reaction (PCR). The unique E. coli chromosomal sequence located between these primer binding sites is amplified by PCR and used as a probe to identify the recombinant clones from the Kohara phage ordered E. coli miniset bank that contains the Tn5 mutated loci. We used this approach to map two Tn5 insertion mutations previously identified by their effect on glycerol metabolism. The insertion mutations mapped to glpD, the aerobic sn-glycerol-3-phosphate dehydrogenase gene. Phenotypic analysis of the mutant strains revealed one with partial GlpD activity, suggesting transposon-mediated alteration of promoter activity. This mapping method should be applicable to the rapid physical mapping of any insertion mutation in the E. coli chromosome.

Dispersed repetitive DNA sequences have been described recently in eubacteria. To assess the distribution and evolutionary conservation of two distinct prokaryotic repetitive elements, consensus oligonucleotides were used in polymerase chain reaction [PCR] amplification and slot blot hybridization experiments with genomic DNA from diverse eubacterial species. Oligonucleotides matching Repetitive Extragenic Palindromic [REP] elements and Enterobacterial Repetitive Intergenic Consensus [ERIC] sequences were synthesized and tested as opposing PCR primers in the amplification of eubacterial genomic DNA. REP and ERIC consensus oligonucleotides produced clearly resolvable bands by agarose gel electrophoresis following PCR amplification. These band patterns provided unambiguous DNA fingerprints of different eubacterial species and strains. Both REP and ERIC probes hybridized preferentially to genomic DNA from Gram-negative enteric bacteria and related species. Widespread distribution of these repetitive DNA elements in the genomes of various microorganisms should enable rapid identification of bacterial species and strains, and be useful for the analysis of prokaryotic genomes.

We undertook clinical evaluation (32 cases) and molecular evaluation (31 cases) of unrelated patients affected with Smith-Magenis syndrome (SMS) associated with an interstitial deletion of band p11.2 of chromosome 17. Patients were evaluated both clinically and electrophysiologically for peripheral neuropathy, since markers showing close linkage to one form of Charcot-Marie-Tooth disease (CMT1A) map to this chromosomal region. The common clinical findings were broad flat midface with brachycephaly, broad nasal bridge, brachydactyly, speech delay, and hoarse, deep voice. Fifty-five percent of the patients showed clinical signs (e.g., decreased or absent deep tendon reflexes, pes planus or pes cavus, decreased sensitivity to pain, and decreased leg muscle mass) suggestive of peripheral neuropathy. However, unlike patients with CMT1A, these patients demonstrated normal nerve conduction velocities. Self-destructive behaviors, primarily onychotillomania and polyembolokoilamania, were observed in 67% of the patients, and significant symptoms of sleep disturbance were observed in 62%. The absence of REM sleep was demonstrated by polysomnography in two patients. Southern analysis indicated that most patients were deleted for five 17p11.2 markers--FG1 (D17S446), 1516 (D17S258), pYNM67-R5 (D17S29), pA10-41 (D17S71), and pS6.1-HB2 (D17S445)--thus defining a region which appears to be critical to SMS. The deletion was determined to be of paternal origin in nine patients and of maternal origin in six patients. The apparent random parental origin of deletion documented in 15 patients suggests that genomic imprinting does not play a role in the expression of the SMS clinical phenotype. Our findings suggest that SMS is likely a contiguous-gene deletion syndrome which comprises characteristic clinical features, developmental delay, clinical signs of peripheral neuropathy, abnormal sleep function, and specific behavioral anomalies.

Duchenne muscular dystrophy is a lethal X-linked recessive disorder caused by the deficiency of a component of the muscle fiber membrane cytoskeleton called dystrophin. Becker muscular dystrophy, a clinically milder disorder, results from dystrophin abnormalities rather than deficiency. We identified the first patient who is clearly an exception to these established clinical and biochemical correlates. The patient described clinically had particularly severe Duchenne dystrophy. Biochemically, his muscle contained substantial amounts of abnormal dystrophin (Becker-like). Characterization of the dystrophin protein and gene revealed a unique intragenic gene deletion resulting in a dystrophin protein missing the carboxyl-terminal domain. This patient's dystrophin seemed to have a deleterious "dominant" effect on his muscle: The presence of this abnormal protein was more damaging to the myofibers than the absence of dystrophin would have been. This patient challenges the current hypothesis that dystrophin associates with the plasma membrane solely via its carboxyl-terminus, yet supports the hypothesis that an intact carboxyl-terminus is crucial for correct dystrophin function.

In 1990, Richards et al. reported dramatically skewed lyonization in a set of female monozygotic twins heterozygous for Duchenne muscular dystrophy (DMD). The skewed inactivation pattern was symmetrical in opposite directions, one twin being affected with DMD, the other one being normal. Here, we report an additional set of female monozygotic twins heterozygous for a mutation at the dystrophin locus. Similarly, one shows a manifesting carrier phenotype while one is normal. However, unlike the previous report, we find a skewed X inactivation pattern only in the affected twin, while the normal twin showed a random X inactivation pattern. Our results lend considerable experimental support for the models of twinning and X inactivation recently outlined by Nance in 1990, in that these twins probably represent asymmetric splitting of the inner cell mass (ICM): The affected twin likely arose when a small proportion of the ICM split off after lyonization had occurred. In this situation, the original ICM could give rise to the normal twin with random lyonization, while the newly split cells would experience catch-up growth and lead to the affected twin. Genetic studies of this family showed that the specific dystrophin gene mutation was an exon duplication that arose sporadically in the paternally derived X chromosome.

We report on an infant, born to a diabetic mother, who presented with hypocalcemia and congenital heart disease, presurgically diagnosed by echocardiography as truncus arteriosus type I. Cytogenetic analysis showed a 45,X,-Y,-22,+der-(Y)t(Y;22) (p11.3q11.2) chromosome abnormality with del(22)(q11.2). Parental chromosomes were normal. Autopsy showed persistent truncus arteriosus type II and thymic aplasia consistent with DiGeorge anomaly. This report adds to the existing literature demonstrating an association between DiGeorge anomaly and monosomy 22q11.

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.

We report a patient (S.T.) with multiple congenital anomalies and developmental delay associated with an interstitial deletion of 1q23-1q25. Molecular analysis of the deletion was performed using DNA markers that map to 1q. Five DNA markers, MLAJ-1 (D1S61), CRI-L1054 (D1S42), HBI40 (D1S66), OS-6 (D1S75), and BH516 (D1S110), were demonstrated to be deleted. Informative polymorphisms demonstrated this to be a de novo deletion of the maternally derived chromosome. Deletion status was determined using restriction fragment length polymorphism (RFLP) analysis supplemented with densitometry in the experiments where RFLP analysis was not fully informative. Deletions were confirmed by Southern analysis using genomic DNA from a somatic cell hybrid retaining the del(1)(q23-q25) chromosome that was constructed from patient S.T. Flow karyotyping confirmed the deletion and estimated that the deletion encompassed 11,000-16,000 kb. The clinical and cytogenetic characteristics of S.T. are compared with those of ten previously described patients with monosomy 1q21-1q25.

Eleven conditional lethal dnaG(Ts) mutations were located by chemical cleavage of heteroduplexes formed between polymerase chain reaction-amplified DNAs from wild-type and mutant dnaG genes. This entailed end labeling one DNA strand of the heteroduplex, chemically modifying the strands with hydroxylamine or osmium tetroxide (OsO4) at the site of mismatch, and cleaving them with piperidine. The cleavage products were electrophoresed, and the size corresponded to the position of the mutation with respect to the labeled primer. Exact base pair changes were then determined by DNA sequence analysis. The dnaG3, dnaG308, and dnaG399 mutations map within 135 nucleotides of one another near the middle of dnaG. The "parB" allele of dnaG is 36 bp from the 3' end of dnaG and 9 bp downstream of dnaG2903; both appear to result in abnormal chromosome partitioning and diffuse nucleoid staining. A suppressor of the dnaG2903 allele (sdgA5) maps within the terminator T1 just 5' to the dnaG gene. Isogenic strains that carried dnaG2903 and did or did not carry the sdgA5 suppressor were analyzed by a combination of phase-contrast and fluorescence microscopy with 4',6-diamidino-2-phenylindole to stain DNA and visualize the partitioning chromosome. Overexpression of the mutant dnaG allele corrected the abnormal diffuse-nucleoid-staining phenotype associated with normally expressed dnaG2903. The mutations within the dnaG gene appear to cluster into two regions which may represent distinct functional domains within the primase protein.

Charcot-Marie-Tooth disease type 1A is a demyelinating, inherited peripheral neuropathy which is associated with a DNA duplication in chromosome 17p11.2-p12 in over 70% of patients with CMT1A. The CMT1A duplication is not detected cytogenetically, and constitutes a tandem duplication of a 1.5-Mb region of DNA flanked by homologous sequences designated as CMT1A-REP. Detection of the CMT1A duplication by molecular methods is a valuable diagnostic test for the majority of CMT1A cases. This duplication mutation shows stable inheritance through multiple generations, and may also arise as a new mutation in sporadic patients. The CMT1A duplication leads to the disease phenotype apparently through increased dosage of a gene(s) within the duplicated segment. A disease gene associated with CMT1A has been identified in the form of PMP22, which maps within the CMT1A duplication region, and encodes a myelin protein of the peripheral nerve. Point mutations in the PMP22 gene have been identified in CMT1A patients, including one case of a new mutation in PMP22 which coincided with the onset of the disease. Thus, two alternative molecular mechanisms are responsible for CMT1A: DNA duplication leading to increased gene dosage, and point mutation of the PMP22 gene.

We demonstrate that the digestion of template DNAs with restriction endonucleases prior to Alu polymerase chain reaction ("restricted Alu-PCR") reduces the complexity of the Alu-primed amplification patterns of human DNA in somatic cell hybrids and allows a direct informative comparison of these patterns. A comparison of restricted Alu-PCR patterns of a monochromosomal hybrid retaining a human chromosome 17 (MH22-6) and a hybrid retaining a human chromosome 17 deleted for band p11.2 (DH110-D1) revealed four Alu-PCR products that were present in the former but absent in the latter hybrid. Hybridization of these fragments to the total Alu-PCR amplification products of the two hybrids confirmed their absence in DH110-D1 amplification products. Hybridization to a panel of somatic cell hybrids indicated that two of these fragments were deleted in the hybrid DH110-D1 and mapped to 17p11.2, as expected. However, two additional fragments were not deleted in the hybrid DH110-D1 and mapped to other regions of chromosome 17. An insertion-deletion polymorphism was associated with one of the latter fragments, which may be the mechanism for the lack of its amplification in the hybrid DH110-D1. Restricted Alu-PCR should enhance the applications of Alu-PCR and provides a new method for the identification of chromosome-specific polymorphic markers.

We report the isolation of a new marker (S6.1) from band p11.2 of human chromosome 17 by differential Alu-polymerase chain reaction (Alu-PCR) of both a monochromosomal hybrid retaining a single human chromosome 17 and a hybrid retaining a del(17)(p11.2p11.2) in addition to other human chromosomes. The method is based on the preferential PCR amplification of human DNA in rodent/human hybrids when primers specific to the human Alu repeat element are used. MspI and SstI RFLPs associated with S6.1 were identified and used in linkage analysis of both a previously reported and a newly identified French-Acadian kindred segregating autosomal dominant Charcot-Marie-Tooth disease (CMT). A cumulative peak lod score of 3.41 at a peak recombination fraction of .12 indicates that this marker is linked to the CMT 1A locus but is at a distance from the disease gene. Thus, the marker S6.1 will be useful in further delineating the candidate region for the CMT gene when its location with respect to pA10-41 and 1516, two other markers from 17p11.2 which have previously demonstrated close linkage to the CMT locus, has been determined.

This report describes the cloning and sequencing of a chromosomally encoded tetracycline resistance determinant from a clinical isolate of methicillin-resistant Staphylococcus aureus. On the basis of the sequence, the gene is in the tet(M) class, and it was shown that the S. aureus tetA(M) gene is induced at the level of transcription.

Transposon Tn5 mutagenesis of the Escherichia coli chromosome was used to isolate 21 independent insertion mutations conferring an altered colony color phenotype on MacConkey-glycerol plates. The polymerase chain reaction was used to map 16 of these Tn5 insertions within the glpFK region at 88 min. The most polar Tn5 insertion was shown by nucleotide sequencing to be in the proposed glpF open reading frame. The data suggest that the glpF and glpK genes are in an operon with a bent DNA segment (BENT-6) involved in transcriptional regulation of this operon.

We have performed linkage analysis in a large French-Acadian kindred segregating one form of autosomal dominant Charcot-Marie-Tooth disease (CMTD) (type IA) using 17 polymorphic DNA markers spanning human chromosome 17 and demonstrate linkage to several markers in the pericentromeric region, including DNA probes pA10-41, EW301, S12-30, pTH17.19, c11-2B, and p11-2c11.5. Linkage of markers pA10-41 and EW301 to CMTD type IA has been reported elsewhere. Four new markers, 1516, 1517, 1541, and LL101, which map to chromosome 17 have been identified. The marker 1516 appears to be closely linked to the CMTD locus on chromosome 17 as demonstrated by a maximum lod score of 3.42 at theta (recombination fraction) = 0. This marker has been mapped to 17p11.2 using a somatic cell hybrid constructed from a patient with Smith-Magenis syndrome [46,XY, del(17)(p11.2p11.2)]. A lod score of 6.16 has been obtained by multipoint linkage analysis with 1516 and two markers from 17q11.2, pTH17.19, and c11-2B. The markers 1517 and 1541 have been mapped to 17p12-17q11.2 and demonstrate maximum lod scores of 2.35 and 0.63 at recombination values of .1 and .2, respectively. The marker LL101 has been mapped to 17p13.105-17p13.100 and demonstrates a maximum lod score of 1.56 at a recombination value of .1. Our study confirms the localization of CMTD type IA to the pericentromeric region of chromosome 17.

A 24 kilobase pair region of the E. coli chromosome surrounding the dnaG gene has been cloned and characterized. A lambda phage library was constructed by ligating a Sau3A( decreases GATC) partial DNA digest of the entire E. coli chromosome into the lambda BamHI(G decreases GATCC) cloning vector charon 28. Partial digestion was performed to generate overlapping chromosomal fragments and to allow one to walk along the chromosome. This library was probed with a nick-translated plasmid (pRRBl) containing the rpoD gene, which maps adjacent to dnaG at 66 min. Four bacteriophages: lambda 3, lambda 4, lambda 5, lambda 6 that hybridized to the probe were isolated from the 2,500 plaques screened. One phage recombinant lambda 4, was shown to contain the dnaG gene. Three recombinant plasmids containing dnaG: pGL444, pGL445, pBS105, were constructed via subcloning of lambda 4 using different restriction of fragments. Plasmids pGL444 and pBS10 5 were subjected to transposon Tn5 mutagenesis and 88 Tn5 inserts into the cloned region were isolated. The location of the Tn5 inserts were mapped by restriction enzyme analysis of the plasmids and the insertion mutations were checked for ability to complement of dnaGts chromosomal marker at nonpermissive 40 degrees C. In this manner a correlated physical and genetic map of dnaG was determined. A large number of Tn5 inserts map to a specific 900 b.p. region which we propose may be involved in the regulation of dnaG gene expression.

Recombinant plasmids carrying one or both ends of the bacteriophage Mu genome were constructed by molecular cloning. Transposable mini-Mu's with selectable markers (ampicillin resistance, kanamycin resistance or the entire lac operon of Escherichia coli) inserted between the Mu ends were also constructed. As a source of lac operon DNA, a pBR322 derivative with a 27 kb insert containing the lac operon was constructed. The plasmids with both ends of Mu (mini-Mu's) conferred full Mu immunity upon the host cells. However, the same mini-Mu's containing kan or lac inserts were defective in immunity. A summary of the construction and physical characterization, including restriction endonuclease cleavage maps and some of the biological properties of the plasmids, is presented.

The regulatory regions for the rpsU-dnaG-rpoD macromolecular synthesis operon have been fused to a structural gene whose product is readily assayed (the Cmr structural gene coding for chloramphenicol acetyl transferase, CAT). The promoters (P1, P2, P3, Pa, Pb, Phs) for the macromolecular synthesis operon have different strengths as shown by their relative abilities to drive expression of the CAT gene. Promoter occlusion by P1 can be demonstrated within this operon. Regions 5kb upstream have a profound effect on operon gene expression. There is a thermoinducible promoter located within the dnaG structural gene. One of the macromolecular synthesis operon promoters is under lexA control. Although the operon structure allows coordinate expression of rpsU, dnaG and rpoD these additional features suggest that expression of individual genes can be independently regulated in response to altered growth conditions.

Bal31 exonuclease deletion analysis and transposon Tn5 mutagenesis of the 5' regulatory region of the rpsU-dnaG-rpoD macromolecular synthesis operon fused to the chloramphenicol acetyltransferase gene (pGLR301) demonstrated that sequences 5' to the operon promoters were not involved in operon transcriptional regulation and that the three tandem promoters P1, P2, and P3 were functionally independent. P2 was the strongest promoter, and P3 was the weakest. P1, P2, and P3 acting in combination appeared to be stronger than the individual promoters.

We have fused the E. coli dnaG 5' regulatory region to the TcR structural gene in the promoter probe plasmids pPV33 and pKK175-6 to demonstrate that a promoter activity is located on a 250 bp SacII-HindIII restriction fragment and that a transcription terminator, previously reported by nucleotide sequence (Smiley et al. 1982) to immediately precede the dnaG gene, acts as such in vivo. We have determined the complete nucleotide sequence of this controlling region and report: 1) tandem promoters on the same SacII-HindIII restriction fragment which promotes tet expression in the gene fusion experiments, 2) an open reading frame between these promoters and the dnaG gene which is the rpsU (ribosomal protein S21) gene, 3) a sequence homologous to the lambda nut site, 4) a possible LexA protein binding site on one of the dnaG promoters. This places the order on the E. coli genetic map at 66 min in the clockwise direction as rpsU-dnaG-rpoD which are all contained in a single macromolecular synthesis operon. We postulate a model for regulation of the initiation of DNA replication based on antitermination of the rpsU-dnaG-rpoD operon.

The nucleotide sequence of a cloned section of the Escherichia coli chromosome containing the dnaG primase gene [Lupski, J., Smiley, B., Blattner, F. & Godson, G. N. (1982) Mol. Gen, Genet. 185, 120--128] has been determined. The region coding for the dnaG primase has been identified by NH2-terminal and tryptic peptide amino acid analysis of the dnaG protein. The coding region is 1,740 base pairs long (580 amino acids) and is preceded by an unusual ribosome-binding site sequence (G-G-G-G). The dnaG gene is read in the same direction as the adjacent rpoD gene, but no obvious promoter sequences can be found for either gene within several hundred nucleotides upstream. Other unusual features of the dnaG gene that may explain the maintenance of its product at low copy number are the presence of a RNA polymerase terminator 31 nucleotides upstream from the ATG initiator codon and greater use (3--10 times) of certain condons that occur infrequently in other E. coli genes. The nucleotide sequence has also been correlated with data from transposon Tn5 insertional inactivation mapping.

A recombinant complementary DNA clone from Plasmodium knowlesi makes a beta-lactamase fusion polypeptide in Escherichia coli that reacts with a monoclonal antibody to a Plasmodium surface antigen. An epitope of the surface antigen was localized by transposon Tn5 mutagenesis mapping of the complementary DNA clone. The Tn5 mutation having the farthest 5' insert into the complementary DNA portion of the chimeric gene, giving the shortest truncated protein that maintained the ability to bind monoclonal antibody, defined the location of the epitope.