The rust transferred proteins—a new family of effector proteins exhibiting protease inhibitor function

Only few fungal effectors have been described to be delivered into the host cell during obligate biotrophic interactions. RTP1p, from the rust fungi Uromyces fabae and U. striatus, was the first fungal protein for which localization within the host cytoplasm could be demonstrated directly. We investigated the occurrence of RTP1 homologues in rust fungi and examined the structural and biochemical characteristics of the corresponding gene products. The analysis of 28 homologues showed that members of the RTP family are most likely to occur ubiquitously in rust fungi and to be specific to the order Pucciniales. Sequence analyses indicated that the structure of the RTPp effectors is bipartite, consisting of a variable N‐terminus and a conserved and structured C‐terminus. The characterization of Uf‐RTP1p mutants showed that four conserved cysteine residues sustain structural stability. Furthermore, the C‐terminal domain exhibits similarities to that of cysteine protease inhibitors, and it was shown that Uf‐RTP1p and Us‐RTP1p are able to inhibit proteolytic activity in Pichia pastoris culture supernatants. We conclude that the RTP1p homologues constitute a rust fungi‐specific family of modular effector proteins comprising an unstructured N‐terminal domain and a structured C‐terminal domain, which exhibit protease inhibitory activity possibly associated with effector function during biotrophic interactions.     

Molecular analysis of two new Steel mutations in mice shows a transversion or an insertion

The nucleotide sequence data reported in this paper have been submitted to the EMBL data base and have been assigned to accession numbers X95379, X95380, X95381, and X99322.     

Genetic mapping of a mouse ocular malformation locus, tcm, to chromosome 4

The Tcm mutation in the mouse is an autosomal dominant ocular malformation manifesting as microphthalmia, iris dysplasia, cataract, and coloboma. As a first step to cloning the Tcm gene, we report the localization of the Tcm mutation with respect to known microsatellite markers. Backcross progeny carrying the Tcm mutation were produced by mating Tcm/+ heterozygous mice to normal C57BL/6 partners. Genomic DNA from each mouse was subjected to PCR analysis to identify simple sequence length polymorphisms. Our results locate Tcm to Chr 4 and suggest candidate genes responsible for the Tcm phenotype. Finally, ocular histopathology was done in 3-week-old animals to define the extent of the malformation. Received: 14 April 1996 / Accepted: 13 October 1996     

Relationship of Pax6 activity levels to the extent of eye development in the mouse, Mus musculus

In this study we extend the mouse Pax6 mutant allelic series to include a homozygous and hemizygous viable hypomorph allele. The Pax6(132-14Neu) allele is a Phe272Ile missense mutation within the third helix of the homeodomain. The mutant Pax6 homeodomain shows greatly reduced binding activity to the P3 DNA binding target. Glucagon-promoter activation by the entire mutant Pax6 product of a reporter gene driven by the G1 paired and homeodomain DNA binding target was slightly increased. We constructed mutant Pax6 genotypes such that Pax6 activity ranged between 100 and 0% and show that the extent of eye development is progressively reduced as Pax6 activity decreased. Two apparent thresholds identify three groups in which the extent of eye development abruptly shifted from complete eye at the highest levels of Pax6 to a rudimentary eye at intermediate levels of Pax6 to very early termination of eye development at the lowest levels of Pax6. Of the two Pax6-positive regions that participate in eye development, the surface ectoderm, which develops into the lens vesicle and the cornea, is more sensitive to reduced levels of Pax6 activity than the optic vesicle, which develops into the inner and outer retinal layers.     

Analysis of Pax6 Contiguous Gene Deletions in the Mouse,Mus musculus,Identifies Regions Distinct from Pax6 Responsible for Extreme Small-Eye and Belly-Spotting Phenotypes

In the mouse Pax6 function is critical in a dose-dependent manner for proper eye development. Pax6 contiguous gene deletions were shown to be homozygous lethal at an early embryonic stage. Heterozygotes express belly spotting and extreme microphthalmia. The eye phenotype is more severe than in heterozygous Pax6 intragenic null mutants, raising the possibility that deletions are functionally different from intragenic null mutations or that a region distinct from Pax6 included in the deletions affects eye phenotype. We recovered and identified the exact regions deleted in three new Pax6 deletions. All are homozygous lethal at an early embryonic stage. None express belly spotting. One expresses extreme microphthalmia and two express the milder eye phenotype similar to Pax6 intragenic null mutants. Analysis of Pax6 expression levels and the major isoforms excluded the hypothesis that the deletions expressing extreme microphthalmia are directly due to the action of Pax6 and functionally different from intragenic null mutations. A region distinct from Pax6 containing eight genes was identified for belly spotting. A second region containing one gene (Rcn1) was identified for the extreme microphthalmia phenotype. Rcn1 is a Ca⁺²-binding protein, resident in the endoplasmic reticulum, participates in the secretory pathway and expressed in the eye. Our results suggest that deletion of Rcn1 directly or indirectly contributes to the eye phenotype in Pax6 contiguous gene deletions.CONTIGUOUS gene deletions account for a significant portion of human genetic syndromes. The application of fluorescence in situ hybridization (FISH) cytogenetics and array comparative genome hybridization (array-CGH) technologies have enabled more accurate localization of deletion breakpoints. This deletion information combined with the annotation of the human genome structure provides critical information to identify genes responsible for particular phenotypes associated with a syndrome. For example, deletions of the 11p11p12 and 11p13 regions on the short arm of human chromosome (Chr) 11 have been identified in the Potocki–Shaffer syndrome (Shaffer et al. 1993; Bartsch et al. 1996; Potocki and Shaffer 1996) and the Wilm''s tumor- aniridia- genitourinary abnormalities- mental retardation (WAGR) syndrome (Riccardi et al. 1978; Francke et al. 1979; Hittner et al. 1979; Fryns et al. 1981), respectively. Deletion analyses were important in identifying genes associated with clinical features of the syndromes: EXT2 for multiple exostoses and ALX4 for parietal foramina in Potocki–Shaffer syndrome (Ligon et al. 1998; Wu et al. 2000; Wakui et al. 2005), WT1 for Wilm''s tumor, and PAX6 for aniridia in WAGR syndrome (van Heyningen et al. 1985; Glaser et al. 1986, 1992; Fantes et al. 1992). Deletion analyses have also defined the extent of the deleted region in patients with combined Potocki–Shaffer and WAGR syndromes (McGaughran et al. 1995; Brémond-Gignac et al. 2005) as well as microdeletions 3′ to PAX6, which prevent expression of PAX6 and cause aniridia (Lauderdale et al. 2000; D''elia et al. 2007; Davis et al. 2008).The mouse Chr 2 region homologous to the human WAGR region contains the genes Wt1, Rcn1, Pax6, and Elp4. An extensive allelic series at Pax6 has been identified (Bult et al. 2008). Heterozygote Pax6 intragenic null mutants express microphthalmia, iris anomalies, corneal opacities, lens opacities, and lens-corneal adhesions. Homozygotes are anophthalmic and die shortly after birth (Roberts 1967; Hogan et al. 1986). Five deletions in the region have been identified: Pax6^Sey-Dey, Pax6^Sey-H, Pax6^Sey-2H, Pax6^Sey-3H, Pax6^Sey-4H of which two, Pax6^Sey-H (Hogan et al. 1986; Kent et al. 1997; Kleinjan et al. 2002; Webb et al. 2008) and Pax6^Sey-Dey (Theiler et al. 1978; Hogan et al. 1987; Glaser et al. 1990), have been well characterized. Heterozygotes for both deletions express belly spotting and a more extreme eye phenotype than that observed for heterozygotes of intragenic Pax6 null mutations. Homozygotes for both deletions are lethal at an early embryonic stage.We were particularly interested in the extreme eye phenotype associated with the Pax6 deletions and considered two alternative hypotheses. Either Pax6 deletions are functionally different from Pax6 intragenic null mutations or deletion of a region linked to but distinct from the Pax6 structural gene affects the eye phenotype.In the present study we identify three new deletions encompassing the Pax6 region of the mouse. They have been assigned the mutant allele symbols Del(2)Pax6^11Neu/1Neu, Del(2)Pax6^12Neu/2Neu, and Del(2)Pax6^13Neu/3Neu and will be referred to throughout this publication as Pax6^11Neu, Pax6^12Neu, and Pax6^13Neu, respectively. All three deletions are homozygous lethal at an early embryonic stage. The deletions differentiate for the extent of the eye abnormality expressed: Pax6^11Neu heterozygotes express extreme microphthalmia similar to that observed in the Pax6^Sey-Dey and Pax6^Sey-H deletions. Pax6^12Neu and Pax6^13Neu heterozygotes express the milder eye abnormality seen in heterozygous intragenic null mutants. For all three deletions, heterozygotes do not express belly spotting. Genetic, phenotypic, and molecular characterization of the deletions allowed us to identify regions associated with the array of phenotypes in these contiguous gene deletions.     

A new pyruvate kinase mutation with hyperactivity in the mouse

A mouse mutant with pyruvate kinase (PK) hyperactivity has been found in offspring of 1-ethyl-1-nitrosourea (ENU)-treated male mice. The activity alteration was detected in the blood and could also be found in the liver but not in the muscle, kidney, heart, spleen, lung, or brain. Heterozygous mice have erythrocyte PK activity enhanced up to about 160% and homozygotes up to about 240%, compared to homozygous wild types. The mutation is codominantly expressed. The heterozygous and homozygous mutants are viable and fully fertile and do not show symptoms of erythrocytosis. The mutation does not affect the heat stability, the electrophoretic mobility, or the Km (for phosphoenolpyruvate) of the PK molecule. It is suggested that the regulatory locus of PK-1 is affected by this mutation. The observations support also the theory of one structural locus for the erythrocyte and liver isozymes.