A fifth locus for primary autosomal recessive microcephaly maps to chromosome 1q31.

Primary microcephaly is a genetic disorder in which an affected individual has a head circumference >3 SDs below the age- and sex-related mean. A small but apparently normally formed brain is the reason for the reduced head circumference, and, probably because of this, all affected individuals are mentally retarded. The condition is genetically heterogeneous, and four loci have already been identified. We now report a fifth locus, MCPH5, which is an 8-cM region mapping to chromosome 1q31, defined by the markers GATA135F02 and D1S1678.

The second locus for autosomal recessive primary microcephaly (MCPH2) maps to chromosome 19q13.1-13.2.

Primary microcephaly is a clinical diagnosis made when an individual has a head circumference of greater than 3 standard deviations below the age and sex matched population mean, mental retardation but without other associated malformations and no apparent aetiology. The majority of cases of primary microcephaly exhibit an autosomal recessive mode of inheritance. We now demonstrate the genetic heterogeneity of this condition with the identification of a second primary microcephaly locus (MCPH2) on chromosome 19q13.1-13.2 in two multi-affected consanguineous families. The minimum critical region containing the MCPH2 locus is defined by the polymorphic markers D19S416 and D19S420 spanning a region of approximately 7.6 cM.

Potassium permanganate and tetraethylammonium chloride are a safe and effective substitute for osmium tetroxide in solid-phase fluorescent chemical cleavage of mismatch.

Whilst chemical cleavage of mismatch (CCM) detects all point mutations in DNA, its widespread use has been hampered by the complex multistage methodology and the need for toxic chemicals, in particular osmium tetroxide. Here we show that osmium tetroxide can be replaced by potassium permanganate, giving the same spectrum of mutation detection, but with greater sensitivity. The use of potassium permanganate is compatible with solid phase capture and fluorescent detection, giving a safer method of mutation detection. We present here a comparison of CCM with osmium tetroxide and with potassium permanganate, tested on a complete set of single base pair mismatches and a number of small insertion/deletions.

Comparison of enzyme mismatch cleavage and chemical cleavage of mismatch on a defined set of heteroduplexes.

Two mutation detection methods based on the cleavage of mismatched heteroduplexes were compared and evaluated. These techniques, chemical cleavage of mismatch (CCM) and enzyme mismatch cleavage (EMC), have the advantages over other available methods of being able to detect and localize mutations in relatively large fragments of DNA (> or = 1 kb). We have constructed clones that enable us to create heteroduplexes of 500 bp, 1 kb, and 1.5 kb and have assessed each of the methods over a range of criteria. Both were able to detect and localize all four types of single-base mismatch and insertion/deletions of 1-5 bp. Whereas EMC was efficient at detection of insertion/deletions in a broad size range of fragments and has the advantage over CCM of using no hazardous chemicals, in our hands it has not been sufficiently robust that we felt confident to consider it for diagnostic use in its present form. CCM using hydroxylamine was efficient over the entire range of fragment sizes tested and using potassium permanganate with tetraethylammonium chloride was efficient up to 1 kb.

Depression of streptomycin production by Streptomyces griseus at elevated growth temperature: studies using gene fusions.

Streptomyces griseus ATCC 12475 fails to produce streptomycin when grown at 34 degrees C or above, although growth is appreciable up to at least 37 degrees C. This depression of streptomycin production at elevated growth temperature is manifest equally in liquid and on solid, and with complex and minimal, media. We report studies with gene fusions of the reporter genes aph or xyIE to restriction fragments containing the streptomycin biosynthesis promoter PstrB1. aph constructs were in high, and xyIE constructs in low, copy number vectors. Two strB1 promoter fragments were used, one requiring activation by the pathway-specific activator StrR of S. griseus, the other reportedly activator independent. PstrB1 expression in the aph constructs in S. griseus and in S. lividans was significantly reduced at 37 degrees C compared to 30 degrees C. Some of this reduction could be explained by lower plasmid copy number at the higher temperature, but strR-dependent expression was clearly temperature controlled. Using the xyIE reporter system, the temperature dependence of PstrB1 expression was confirmed but, surprisingly, the strR dependence of the two promoter fragments differed from that observed in the multicopy aph constructs. These data identify a temperature-dependent promoter which may contribute to the depressive effect of elevated growth temperature on streptomycin production.

Dependence on reporter gene of apparent activity in gene fusions of a Streptomyces griseus streptomycin biosynthesis promoter.

The adjacent genes strR-strA-strB1 lie within the large cluster of genes of streptomycin biosynthesis and resistance in Streptomyces griseus. strR encodes a pathway-specific activator StrR, suggested by previous work to be either an antiterminator or a conventional activator, binding to its DNA target via a helix-turn-helix motif. strB1 is transcribed in an StrR-dependent fashion from a promoter (PstrB1) that lies downstream from strA; between PstrB1 and strB1 there is a 300-bp leader region containing numerous inverted repeats that could represent modulatable transcription termination sites. Hybrid plasmids were constructed in vitro with transcriptional fusions in which fragments containing PstrB1 and either the entire leader region ("long" fragments) or a small part of it (the "short" fragment) were cloned upstream of (i) aph as reporter gene, in a high copy number plasmid background, or (ii) xylE as reporter gene, in a low copy number plasmid background. The short fragment directed high levels of APH (aminoglycoside 3'-phosphotransferase) whether StrR was present or not, while the long fragments did not do so in the absence of StrR; one long fragment directed high levels in wild-type S. griseus, in which StrR would be present. Insertion of an extraneous fragment into PstrB1 in the short fragment construct led to loss of APH activity, demonstrating that no adventitious promoter had been formed in the short construct. In vitro deletion of part of the leader region in a long fragment construct led to high APH expression with or without StrR present. Although these results are consistent with the target of StrR being within the leader region, and thus with an antiterminator role, it was found that both long and short fragments in the low copy number background failed to direct high expression of catechol oxygenase (the product of xylE) unless strR was also present on a compatible plasmid. Transfer of PstrB1-xylE fragments to the high copy number vector did not increase catechol oxygenase expression. We interpret these results in terms of an effect, in the hybrid constructs, of one of the reporter genes on promoter function, possibly by affecting local DNA topology.