The scl gene product is required for the generation of all hematopoietic lineages in the adult mouse.

Homozygosity for a null mutation in the scl gene causes mid-gestational embryonic lethality in the mouse due to failure of development of primitive hematopoiesis. Whilst this observation established the role of the scl gene product in primitive hematopoiesis, the death of the scl null embryos precluded analysis of the role of scl in later hematopoietic development. To address this question, we created embryonic stem cell lines with a homozygous null mutation of the scl gene (scl-/-) and used these lines to derive chimeric mice. Analysis of the chimeric mice demonstrates that the scl-/- embryonic stem cells make a substantial contribution to all non-hematopoietic tissues but do not contribute to any hematopoietic lineage. These observations reveal a crucial role for the scl gene product in definitive hematopoiesis. In addition, in vitro differentiation assays with scl-/- embryonic stem cells showed that the scl gene product was also required for formation of hematopoietic cells in this system.     

Development ofgusA reporter gene constructs for cereal transformation: Availability of plant transformation vectors from the CAMBIA Molecular Genetic Resource Service

The use of reporter genes to characterise sequence elements that act to regulate gene expression in transgenic plants has been vital to the development of foreign gene expression strategies for use in cereal transformation. ThegusA locus ofEscherichia coli, which encodes the enzyme-glucuronidase (GUS), is by far the most popular reporter gene used in plant transformation. In this paper we extend the utility of the GUS reporter gene system in cereal transformation by describing and evaluating a number of novel constructs suitable for use in direct gene transfer experiments. These plasmids are all available from the Molecular Genetic Resource Service of the Center for the Application of Molecular Biology to International Agriculture.     

Evaluation of mass spectrometric techniques for charaterization of engineered proteins

A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described. The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation. The various aspects of the procedure and its application is described in detail.     

Locus Heterogeneity for Waardenburg Syndrome is Predictive of Clinical Subtypes

Waardenburg syndrome (WS) is a dominantly inherited and clinically variable syndrome of deafness, pigmentary changes, and distinctive facial features. Clinically, WS type I (WS1) is differentiated from WS type II (WS2) by the high frequency of dystopia canthorum in the family. In some families, WS is caused by mutations in the PAX3 gene on chromosome 2q. We have typed microsatellite markers within and flanking PAX3 in 41 WS1 kindreds and 26 WS2 kindreds in order to estimate the proportion of families with probable mutations in PAX3 and to study the relationship between phenotypic and genotypic heterogeneity. Evaluation of heterogeneity in location scores obtained by multilocus analysis indicated that WS is linked to PAX3 in 60% of all WS families and in 100% of WS1 families. None of the WS2 families were linked. In those families in which equivocal lod scores (between −2 and +1) were found, PAX3 mutations have been identified in 5 of the 15 WS1 families but in none of the 4 WS2 families. Although preliminary studies do not suggest any association between the phenotype and the molecular pathology in 20 families with known PAX3 mutations and in four patients with chromosomal abnormalities in the vicinity of PAX3, the presence of dystopia in multiple family members is a reliable indicator for identifying families likely to have a defect in PAX3.     

Evolutionary relationships of bacterial and archaeal glutamine synthetase genes

Glutamine synthetase (GS), an essential enzyme in ammonia assimilation and glutamine biosynthesis, has three distinctive types: GSI, GSII and GSIII. Genes for GSI have been found only in bacteria (eubacteria) and archaea (archaebacteria), while GSII genes only occur in eukaryotes and a few soil-dwelling bacteria. GSIII genes have been found in only a few bacterial species. Recently, it has been suggested that several lateral gene transfers of archaeal GSI genes to bacteria may have occurred. In order to study the evolution of GS, we cloned and sequenced GSI genes from two divergent archaeal species: the extreme thermophile Pyrococcus furiosus and the extreme halophile Haloferax volcanii. Our phylogenetic analysis, which included most available GS sequences, revealed two significant prokaryotic GSI subdivisions: GSI-a and GSI-. GSIa-genes are found in the thermophilic bacterium, Thermotoga maritima, the low G+C Gram-positive bacteria, and the Euryarchaeota (includes methanogens, halophiles, and some thermophiles). GSI--type genes occur in all other bacteria. GSI-- and GSI--type genes also differ with respect to a specific 25-amino-acid insertion and adenylylation control of GS enzyme activity, both absent in the former but present in the latter. Cyanobacterial genes lack adenylylation regulation of GS and may have secondarily lost it. The GSI gene of Sulfolobus solfataricus, a member of the Crenarchaeota (extreme thermophiles), is exceptional and could not be definitely placed in either subdivision. The S. solfataricus GSI gene has a shorter GSI--type insertion, but like GSI-a-type genes, lacks conserved sequences about the adenylylation site. We suspect that the similarity of GSI- genes from Euryarchaeota and several bacterial species does not reflect a common phylogeny but rather lateral transmission between archaea and bacteria.Correspondence to: J.R. Brown 1073     

Chloroplast targeting of spectinomycin adenyltransferase provides a cell-autonomous marker for monitoring transposon excision in tomato and tobacco

Antibiotic resistance genes can act as either cell autonomous or non-cell autonomous genetic markers with which to monitor the excision of plant transposons. To convert spectinomycin resistance from a noncell autonomous resistance to cell autonomous resistance, a transit peptide for chloroplast localization from a petunia ribulose bisphosphate carboxylase (rbcS) gene was fused in-frame to the aadA gene, which confers spectinomycin and streptomycin resistance. Constructs were generated in which the expression of this chimeric gene was prevented by the presence, in the 5 untranslated leader, of the maize transposons Activator (Ac) or Dissociation (Ds). When progeny of tobacco or tomato plants transformed with these constructs were germinated on spectinomycin-containing medium, germinally revertant and somatically variegated individuals could be distinguished.     

Genetic diversity in and phylogenetic relationships of the Brazilian endemic cotton,Gossypium mustelinum (Malvaceae)

Gossypium mustelinum, one of five tetraploid species in the cotton genus, is geographically restricted to a few states in NE Brazil. Allozyme analysis was used to assess levels and patterns of genetic diversity inG. mustelinum and its relationship to the other tetraploid species. Genetic variation was low, with only 6 of 50 loci examined being polymorphic, a mean of 1.14 alleles per locus and a mean panmictic heterozygosity of 0.08. These estimates are low relative to other tetraploid cotton species, but are typical of island endemics. Interpopulational genetic identities were uniformly high, lending support to the concept of there being only one wild species of Brazilian cotton. The limited allelic diversity observed was correlated with geographical distribution, although variability is so limited in the species that geographically marginal populations are electrophoretically ordinary. Phylogenetic and phenetic analyses demonstrate thatG. mustelinum is isolated among polyploid cotton species, occupying one of the three basal clades resulting from an early radiation of polyploid taxa subsequent to polyploid formation. We suggest thatG. mustelinum represents a paleoendemic that presently exists as a series of widely scattered, relictual populations. Despite several centuries of sympatric cultivation ofG. barbadense andG. hirsutum, there was little evidence of interspecific introgression of alleles from cultivated cottons intoG. mustelinum.     

Analysis of the frequency of inheritance of transposed Ds elements in Arabidopsis after activation by a CaMV 35S promoter fusion to the Ac transposase gene

The Ac/Ds transposon system of maize shows low activity in Arabidopsis. However, fusion of the CaMV 35S promoter to the transposase gene (35S::TPase) increases the abundance of the single Ac mRNA encoded by Ac and increases the frequency of Ds excision. In the experiments reported here it is examined whether this high excision frequency is associated with efficient re-insertion of the transposon. This was measured by using a Ds that carried a hygromycin resistance gene (HPT) and was inserted within a streptomycin resistance gene (SPT). Excision of Ds therefore gives rise to streptomycin resistance, while hygromycin resistance is associated with the presence of a transposed Ds or with retention of the element at its original location. Self-fertilisation of most individuals heterozygous for Ds and 35S::TPase produced many streptomycin-resistant (strep^r) progeny, but in many of these families a small proportion of strep^r seedlings were also resistant to hygromycin (hyg^r). Nevertheless, 70% of families tested did give rise to at least one strep^r, hyg^r seedling, and over 90% of these individuals carried a transposed Ds. In contrast, the Ac promoter fusion to the transposase gene (Ac::TPase) produced fewer strep^rhyg^r progeny, and only 53% of these carried a transposed Ds. However, a higher proportion of the strep^r seedlings were also hyg^r than after activation by 35S::TPase. We also examined the genotype of strep^r, hyg^r seedlings and demonstrated that after activation by 35S::TPase many of these were homozygous for the transposed Ds, while this did not occur after activation by Ac::TPase. From these and other data we conclude that excisions driven by 35S::TPase usually occur prior to floral development, and that although a low proportion of strep^r progeny plants inherit a transposed Ds, those that do can be efficiently selected with an antibiotic resistance gene contained within the element. Our data have important implications for transposon tagging strategies in transgenic plants and these are discussed.