Molecular characterization of Pax6(2Neu) through Pax6(10Neu): an extension of the Pax6 allelic series and the identification of two possible hypomorph alleles in the mouse Mus musculus.

Phenotype-based mutagenesis experiments will increase the mouse mutant resource, generating mutations at previously unmarked loci as well as extending the allelic series at known loci. Mapping, molecular characterization, and phenotypic analysis of nine independent Pax6 mutations of the mouse recovered in mutagenesis experiments is presented. Seven mutations result in premature termination of translation and all express phenotypes characteristic of null alleles, suggesting that Pax6 function requires all domains to be intact. Of major interest is the identification of two possible hypomorph mutations: Heterozygotes express less severe phenotypes and homozygotes develop rudimentary eyes and nasal processes and survive up to 36 hr after birth. Pax6(4Neu) results in an amino acid substitution within the third helix of the homeodomain. Three-dimensional modeling indicates that the amino acid substitution interrupts the homeodomain recognition alpha-helix, which is critical for DNA binding. Whereas cooperative dimer binding of the mutant homeodomain to a paired-class DNA target sequence was eliminated, weak monomer binding was observed. Thus, a residual function of the mutated homeodomain may explain the hypomorphic nature of the Pax6(4Neu) allele. Pax6(7Neu) is a base pair substitution in the Kozak sequence and results in a reduced level of Pax6 translation product. The Pax6(4Neu) and Pax6(7Neu) alleles may be very useful for gene-dosage studies.

Molecular, genetic and biochemical characterization of lactate dehydrogenase-A enzyme activity mutations in Mus musculus.

Four independent heterozygous lactate dehydrogenase (LDH) mutations with approximately 60% of wild-type enzyme activity in whole blood have been recovered. The mutant line Ldh1a2Neu proved to be homozygous lethal, whereas for the three lines Ldh1a7Neu, Ldh1a11Neu, and Ldh1a12Neu homozygous mutants with about 20% residual activity occurred in the progeny of heterozygous inter se matings. However, the number of homozygous mutants was less than expected, suggesting an increased lethality of these animals. Various physicochemical and kinetic properties of LDH are altered. Exons of the Ldh1 gene were PCR amplified and sequenced to determine the molecular lesion in the mutant alleles. Ldh1a2Neu carried an A/T-->G/C transition in codon 112 (in exon 3), resulting in an Asn-->Asp substitution; Asn112 is part of the helix alpha D, which is involved in the coenzyme-binding domain. Ldh1a7Neu contained an A/T-->C/G transversion within the codon for residue 194 in exon 4, causing an Asp-->Ala substitution, which may affect the arrangement of the substrate-binding site. Three base substituions were discovered for the mutation Ldh1a11Neu in exon 7: the transition C/G-->T/A, a silent mutation, and two transversions C/G-->A/T and C/G-->G/C, both missense mutations, which led to the amino acid replacements A1a319-->Glu and Thr321-->Ser, respectively, located in the alpha H helix structure of the COOH tail of LDHA. We suggest that the mutation in the result of a gene conversion event between Ldh1a wild-type gene and the pseudogene Ldhl-ps. The alteration Ile-->Thr of codon 241 in exon 6 caused by the base pair change T/A-->C/G was identified in the mutation Ldh1a12Neu; Ile241 is included in the helix alpha 2G, a structure that is indirectly involved in coenzyme binding. Each of the sequence alterations has a potential impact on the structure of the LDHA protein, which is consistent with the decreased LDH activity and biochemical and physiological alterations.

Aphakia (ak), a mouse mutation affecting early eye development: fine mapping, consideration of candidate genes and altered Pax6 and Six3 gene expression pattern.

The homozygous mouse mutant aphakia (ak) has been characterized by bilaterally aphakic eyes without a pupil [Varnum DS, Stevens, LC (1968): J Hered 59:147-150]. The mutation was mapped to chromosome 19 [Varnum DS, Stevens, LC (1975): Mouse News Lett 53:35]. Our linkage studies yielded a precise localization of the ak gene 0.6 +/- 0.3 cM proximal to the microsatellite marker D19Mit10 and 0.7 +/- 0.4 cM distal to D19Mit4 and D19Mit91. No recombination was found with the marker D19Mit9 among 418 backcross offspring tested. The developmental control gene Pax2 mapped 11.0 +/- 3.5 cM proximal to ak and is excluded as a candidate gene. Sequence analysis of Fgf8 and Chuk1, which are localized close to the marker D19Mit10, detected no mutations in the ak/ak mutants. Histological analysis of homozygous mutants suggested the arrest of lens development at the lens stalk stage, a transient morphological structure during the formation of the lens vesicle. In the lens remnants, Pax6 and Six3 are expressed, whereas in the persisting lens stalk only Pax6 was detected. The expression pattern of Pax2 appeared normal; Cryaa expression could not be detected. As a consequence of the arrested lens development, other ocular tissues that require for their development information from the intact lens, such as iris, ciliary muscle, retina, and vitreous body, are absent or formed abnormally.

The mouse Pax2(1Neu) mutation is identical to a human PAX2 mutation in a family with renal-coloboma syndrome and results in developmental defects of the brain, ear, eye, and kidney.

We describe a new mouse frameshift mutation (Pax2(1Neu)) with a 1-bp insertion in the Pax2 gene. This mutation is identical to a previously described mutation in a human family with renal-coloboma syndrome [Sanyanusin, P., McNoe, L. A., Sullivan, M. J., Weaver, R. G. & Eccles, M. R. (1995) Hum. Mol. Genet. 4, 2183-2184]. Heterozygous mutant mice exhibit defects in the kidney, the optic nerve, and retinal layer of the eye, and in homozygous mutant embryos, development of the optic nerve, metanephric kidney, and ventral regions of the inner ear is severely affected. In addition, we observe a deletion of the cerebellum and the posterior mesencephalon in homozygous mutant embryos demonstrating that, in contrast to mutations in Pax5, which is also expressed early in the mid-hindbrain region, loss of Pax2 gene function alone results in the early loss of the mid-hindbrain region. The mid-hindbrain phenotype is similar to Wnt1 and En1 mutant phenotypes, suggesting the conservation of gene regulatory networks between vertebrates and Drosophila.

Molecular analysis of four lactate dehydrogenase-A mutants in the mouse.

Four electrophoretic and/or enzyme-activity variants of murine LDH-A subunit (Ldhla-m1Neu, Ldhla-m5Neu, Ldhla-m6Neu, Ldhla-m9Neu), induced by procarbazine hydrochloride or ethylnitrosourea (ENU), were analyzed at the DNA level. The exons of the Ldhl gene from homozygous mutants were amplified by PCR and sequenced. Three mutations resulted from nucleotide substitutions in exon 5: the transitions A-->G at codons 216 (Ldhla-m5Neu) and 225 (Ldhla-m6Neu), and the transversion G-->C (Ldhla-m1Neu) at codon 222. The mutations resulted in the replacements of Glu by Gly (Ldhla-m5Neu), Gln by Arg (Ldhla-m6Neu) and Asp by His (Ldhla-m1Neu). The fourth base substitution, the transition T-->C (Ldhla-m9Neu), has been found at the GT donor splice site following the first exon; this mutation affected the efficiency of transcription. All ENU-induced mutations were A/T-->G/C transitions. The mutation events could be correlated with the biochemical and physiological alterations observed in affected mice.

Close linkage of the dominant cataract mutations (Cat-2) with Idh-1 and cryge on mouse chromosome 1.

The murine dominant gene Cat-2 was located on chromosome 1 between the loci of fuzzy and leaden. Subsequent linkage analysis revealed one recombinant between Cat-2t and isocitrate dehydrogenase-1, and one between Cat-2t and gamma E-crystallin among 338 offspring in three-point backcrosses. The resulting genetic distance between the loci is 0.3 +/- 0.3 cM. The very close linkage between the Cat-2 and the gamma-crystallin gene cluster together with the finding of reduced gamma-crystallin transcripts in mutant lenses suggest strongly that the gamma-crystallin genes may be candidate genes for the Cat-2 mutations.

Genetic instability at the agouti locus of the mouse (Mus musculus). I. Increased reverse mutation frequency to the Aw allele in A/a heterozygotes.

We have compiled the reverse mutation rate data to the white bellied agouti (Aw) allele in heterozygous A/a mice and shown it to be increased by a factor of at least 350 in comparison to the reverse mutation rate in homozygous a/a mice. Employing tightly linked flanking restriction fragment length polymorphism DNA markers, we have shown that reversion to Aw is associated with crossing over in the vicinity of the agouti locus. The non-agouti (a) allele has been recently shown to contain an 11-kb insert within the first intron of the agouti gene. Together with our present results, these observations suggest possible mechanisms to explain the reversion events.

Mutations in the cyclic AMP binding site of the cyclic AMP receptor protein of Escherichia coli.

Mutants in the cyclic AMP binding site of the cyclic AMP receptor protein (CRP) of Escherichia coli have been constructed by oligonucleotide-directed mutagenesis. They have been phenotypically characterized and their ability to enhance the expression of catabolite-repressible operons has been tested. In addition, the binding of cyclic nucleotides to the mutants has been investigated. It is shown that the six mutants made fall into one of three classes: (i) those that bind cyclic AMP better than the wild type protein (Ser-62----Ala) and result in greater transcription enhancement; (ii) those that bind cyclic AMP similarly to wild type (Ser-83----Ala, Ser-83----Lys, Thr-127----Ala, Ser-129----Ala); and (iii) those that do not bind cyclic AMP at all (Arg-82----Leu). Implications of these findings with respect to present models of the cyclic nucleotide binding pocket of CRP are discussed.

Site-directed mutants of the cAMP receptor protein--DNA binding of five mutant proteins.

Oligonucleotide-directed mutagenesis was employed to generate mutants of the cAMP receptor protein (CRP) of Escherichia coli. The mutant proteins were purified to homogeneity and tested for stability and DNA binding. It is shown that mutations at the position of Arg180 abolish specific DNA binding, whereas those at the position Arg185 have very little effect. Both positions have previously been implicated as crucial for the specific interaction between CRP and DNA. The Ser128----Ala mutant shows a slight reduction in DNA binding affinity relative to wild-type. All mutants investigated show similar stability profiles to wild-type CRP with respect to thermolysin proteolysis as a function of temperature.

Structure of the core oligosaccharide from lipopolysaccharide of Erwinia carotovora.

The lipopolysaccharide of Erwinia carotovora was analyzed by quantitative sugar analysis, methylation analysis, and chromic oxide oxidation. This led to the following structure of the core oligosaccharide: (Formula; see text).

Cell wall receptor for bacteriophage Mu G(+).

The invertible G segment in phage Mu DNA controls the host range of the phage. Depending on the orientation of the G segment, two types of phage particles, G(+) and G(-), are produced which recognize different cell surface receptors. The receptor for Mu G(+) was located in the lipopolysaccharide (LPS) of gram-negative bacteria. The analysis of different LPS core types and of mutants that were made resistant to Mu G(+) shows that the primary receptor site on Escherichia coli K-12 lies in the GlcNAc beta 1 . . . 6Glc alpha 1-2Glc alpha 1-part at the outer end of the LPS. Mu shares this receptor site in E. coli K-12 with the unrelated single-stranded DNA phage St-1. Phage D108, which is related to Mu, and phages P1 and P7, which are unrelated to Mu but contain a homologous invertible DNA segment, have different receptor requirements. Since they also bind to terminal glucose in a different configuration, they adsorb to and infect E. coli K-12 strains with an incomplete LPS core.

Immunochemical studies on a Yersinia enterocolitica O:9 lipopolysaccharide cross-reacting with Brucella abortus and Vibrio cholerae extracts.

The presence of two distinct lipopolysaccharides in Yersinia enterocolitica O:9 is described: one isolated from the aqueous phase and one from the phenol phase (Westphal system). The sugar moiety of the phenol phase lipopolysaccharide has been identified as being responsible for the serologic cross-reaction of Y. enterocolitica O:9, Brucella abortus and Vibrio cholerae. The phenol phase antigen consists of glucose, galactose, glucosamine, 2-keto-3-deoxyoctonic acid, heptose, lipid A and a protein moiety. Haemagglutination experiments revealed two different structures: one that readily coats erythrocytes and another that requires alkali treatment. The former may be separated by repeated adsorptions on erythrocytes. Serologically, the two structures behave like a single antigen. The action of normal rabbit serum on the erythrocyte-containg capacity of the two structures was studied.

Biochemical basis of the serological cross-reactions between Brucella abortus and Yersinia enterocolitica serotype O:9.

A method based on the inhibition of agglutination is described that may be used for the differential serological diagnosis between B. abortus and Y. enterocolitica serotype O:9. An antigen with high immunological capacity was isolated from Brucella. This antigen inhibited both homologous and heterologous agglutination by Brucella antiserum, but only the heterologous agglutination by Yersinia antiserum. It proved to be constitued of a polysaccharide (N-acetylglucosamine, glucose, mannose and 2-keto-3-deoxyoctonic acid), a protein and a phosphoglycerid moiety. Lipid A was absent from the Brucella antigen. Incomplete polysaccharide synthesis of the Brucella antigen, with concomitant loss of serological specificity by the rough mutant has been described. Oligosaccharides containing N-acetylgalactosamine, glucose and galactose were isolated from the specific side chain of Yersinia lipopolysaccharide. Lipid A constituents were also identified in the latter.