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Recent results from several laboratories suggest that the interaction of E2 ubiquitin-conjugating enzymes with the RING finger domain has a central role in mediating the transfer of ubiquitin to proteins. Here we present a mutational analysis of the interaction between the E2 enzyme UbcM4/UbcH7 and three different RING finger proteins, termed UIPs, which, like Parkin, contain a RING1-IBR-RING2 motif. The results show that the E2 enzyme binds to the RING1 domain but not to the other cysteine/histidine-rich domains of the RING1-IBR-RING2 motif. Three regions within the UbcM4 molecule are involved in this interaction: the H1 alpha helix, loop L1, connecting the third and fourth strand of the beta sheet, and loop L2, located between the fourth beta strand and the second alpha helix. Loop L2 plays an important role in determining the specificity of interaction. The effects of L2 mutations on UbcM4/UIP interaction are different for each UIP, indicating that RING finger domains can vary considerably in their structural requirements for binding to E2 enzymes. The result that single amino-acid changes can regulate binding of E2 enzymes to different RING finger proteins suggests a novel approach to experimentally manipulate proteolytic pathways mediated by RING finger proteins.

The U-box domain has been suggested to be a modified RING finger motif where the metal-coordinating cysteines and histidines have been replaced with other amino acids. Known U-box-containing proteins have been implicated in the ubiquitin/proteasome system. In a search for proteins interacting with the ubiquitin-conjugating enzyme UbcM4/UbcH7, we have identified a novel U-box containing protein, termed UIP5, that is exclusively found in the nucleus as part of a nuclear dot-like structure. Interaction between UbcM4 and UIP5 was observed in vivo and in vitro with bacterially expressed proteins. In addition to UbcM4, several other ubiquitin-conjugating enzymes (E2s) that share the same sequence within the L1 loop bind to UIP5. Mutational analysis showed that the U-box, like the RING finger in other proteins, forms the physical basis for the interaction with E2 enzymes. Further support for the structural similarity between U-box and RING finger comes from the observation that, in both cases, the same regions within the UbcM4 molecule are required for interaction. Our results establish at the molecular level a link between the U-box and the ubiquitin conjugating system and strongly suggest that proteins containing U-box domains are functionally closely related to RING finger proteins.

The ubiquitin-conjugating enzyme UbcM4, which is identical to the human enzyme UbcH7, was previously shown to be essential for normal mouse development. In order to study the possible role of UbcM4 for cell proliferation and in vitro differentiation, we here describe the establishment and characterization of fibroblast and embryonic stem cell lines with partial or complete inactivation of the UbcM4 gene. ES cell lines in which both alleles of the gene were inactivated by targeted mutagenesis showed no differences in growth rates, cell cycle progression and in vitro differentiation when compared to wild-type ES cells. Fibroblast cell lines with a partially inactivated UbcM4 gene were derived from embryos of the previously described A6 mouse mutant, where retrovirus integration has resulted in a recessive lethal mutation. As in the mutant embryos, steady levels of RNA and protein in the cell lines were reduced by about 70%. The mutant cell lines showed no differences in immortalization kinetics, growth rates and cell cycle progression when compared to wild-type fibroblasts. Taken together, our results strongly suggest that UbcM4-mediated ubiquitination and degradation are not necessary for proteins involved in the maintenance and growth of cells.

CaM kinase II constitutes a family of multifunctional protein kinases that play a major role in Ca2+-mediated signal transduction. As a first step in understanding their possible function in mouse development we characterized the expression patterns of all CaM kinase II isoforms (alpha, beta, gamma and delta) starting in prenatal development. Remarkably, only the ubiquitous gamma- and delta-CaM kinase II are expressed during early development. Their distribution suggests a special role in the developing nervous system and in mature excitable tissues. Additionally, we describe the murine betaM-CaM kinase II, a variant of the 'brain-specific' beta-CaM kinase II, which is highly expressed in skeletal muscle., (Copyright 1999 Elsevier Science B.V.)

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is present in a membrane-bound form that phosphorylates synapsin I on neuronal synaptic vesicles and the ryanodine receptor at skeletal muscle sarcoplasmic reticulum (SR), but it is unclear how this soluble enzyme is targeted to membranes. We demonstrate that alphaKAP, a non-kinase protein encoded by a gene within the gene of alpha-CaM kinase II, can target the CaM kinase II holoenzyme to the SR membrane. Our results indicate that alphaKAP (i) is anchored to the membrane via its N-terminal hydrophobic domain, (ii) can co-assemble with catalytically competent CaM kinase II isoforms and target them to the membrane regardless of their state of activation, and (iii) is co-localized and associated with rat skeletal muscle CaM kinase II in vivo. alphaKAP is therefore the first demonstrated anchoring protein for CaM kinase II. CaM kinase II assembled with alphaKAP retains normal enzymatic activity and the ability to become Ca2+-independent following autophosphorylation. A new variant of beta-CaM kinase II, termed betaM-CaM kinase II, is one of the predominant CaM kinase II isoforms associated with alphaKAP in skeletal muscle SR.

Cell type-specific differences in the transcriptional control of the mouse gene coding for the alpha 1 chain of collagen type I (Col1a1) have been revealed previously with the help of the Mov13 mouse strain which carries a retroviral insert in the first intron of the gene. Transcription of this mutant Col1a1 allele is completely blocked in all mesodermal cell types tested so far, with the exception of the odontoblast where it is expressed at an apparently normal rate (Kratochwil et al. [1989] Cell 57:807-816). To define the tissue specificity of the mutant allele more precisely, we have now studied its expression in osteoblasts, another skeletogenic cell type which, like odontoblasts, produces high amounts of collagen I. Evidence for transcription of the Mov13 allele was obtained by in situ hybridization in homozygous (M/M) and heterozygous (M/+) bone tissue, in grafts as well as in vivo. The presence of mouse collagen I and the development of bone tissue were demonstrated in M/M skeletal elements grown on the chick chorioallantoic membrane (CAM). Further support for expression of the mutant gene was obtained from two 16 day M/M fetuses in vivo. Bone tissue of diverse embryological origin (vertebrae and ribs of somitic origin, long bones derived from lateral plate, calvariae from head paraxial mesoderm, and mandibulae from head neural crest) expresses the mutant allele. However, in situ hybridization experiments indicate that only a subpopulation of osteoblasts is capable of transcribing it at a high rate, resulting in severe impairment of bone development in grafts and in vivo. Therefore, osteoblasts, in comparison to odontoblasts and fibroblast-like cells, assume an intermediate position with respect to transcription of the Mov13 allele. We suggest that this diversity in the utilization of the mutant collagen gene reflects cell type-specific differences in the transcriptional regulation of the wild type (wt) Col1a1 gene.

By exposing preimplantation embryos to Moloney leukemia virus (M-MuLV), we have previously derived substrains of mice designated as Mov-1-Mov-13 which genetically transmit the virus from one generation to the next. In some of the substrains the inserted viral genome becomes activated at specific stages of embryogenesis and the available evidence suggests that these viral genomes are developmentally regulated. To investigate the effect of cellular differentiation on virus expression, M-MuLV was introduced either into preimplantation or post-implantation mouse embryos or into embryonal carcinoma (EC) cells. Whereas preimplantation embryos or EC cells are not permissive for virus expression, efficient replication occurred in postimplantation embryos or in differentiated cell lines. The viral genomes introduced into early embryonal cells were highly methylated and noninfectious when analyzed in the adult. In contrast, viral genomes introduced into postimplantation embryos or into differentiated cells remained unmethylated and were infectious in a transfection assay. These results demonstrate an efficient de novo methylation activity which appears to be involved in repression of genes introduced into pluripotent embryonal cells and which is not observed in cells of the postimplantation embryo or in differentiated cells in tissue culture.

The base sequence of mouse L-cell satellite DNA was investigated by degradation of the two separated complementary strands with the base specific enzyme, T4 endonuclease IV. Digestion of the heavy strand DNA released a limited number of oligonucleotides which were separated by ionophoresis/homochromatography, isolated, and sequenced by the 'wandering spot' method. The light strand DNA was resistant to digestion with T4 endonuclease IV and no detectable amounts of oligonucleotides were released. The oligonucleotides obtained from the heavy strand were related in sequence, indicating that mouse satellite DNA derived from a short tandemly repeated sequence. The sequence of part of the original repeat unit is proposed to be C-A-T-T-T-T-T-C. Five major oligonucleotides were identified, all of which differ from the proposed original sequence by single base changes. The five major oligonucleotides occur with about equal frequency and together comprise approximately 50% of the oligonucleotides released by T4 endonuclease IV from the heavy strand DNA. In addition to the five major oligonucleotides, several oligonucleotides were found to occur in lesser amounts. Since these oligonucleotides are related to the major oligonucleotides, it is likely that they have arisen from them by mutation.

The mammalian X and Y chromosomes, in contrast to the autosomes, pair during male meiosis only near the telomeres. Alleles localized in this region can undergo reciprocal exchange during meiosis. Because such sequences do not show strict sex-linked inheritance, they have been termed pseudoautosomal. In man, several DNA sequences have been described which show pseudoautosomal transmission and which are localized in the pairing region at the ends of the short arms of both the X and Y chromosomes (refs 6-9, and D. Page, unpublished results). We now show that the transgenic mouse strain, Mov-15, contains a single Moloney murine leukaemia virus (M-MuLV) genome in its germline, and genetic evidence indicates that the provirus is integrated into the pseudoautosomal region of the sex chromosome. Proviral copies are lost or gained in 7% of male meioses in this strain, and mouse sequences flanking the provirus are tandemly repeated and highly variable. We conclude that unequal recombination events occur with high frequency in the pairing region, possibly because of the presence of repeated sequences.

The Mov-7 and Mov-9 substrains of mice, carrying Moloney murine leukemia virus (M-MuLV) in their germ line at the Mov-7 locus and Mov-9 locus, respectively, are different with respect to virus activation. Infectious virus appears in all mice carrying the Mov-9 locus but is not activated in animals carrying the Mov-7 locus. Consequently, only Mov-9 mice develop viremia and subsequent leukemia. The endogenous M-MuLV provirus with flanking mouse sequences corresponding to the Mov-7 and Mov-9 loci was molecularly cloned. Detailed restriction maps obtained from the cloned DNAs revealed no detectable differences in the proviral genomes. The flanking mouse sequences, however, were different, confirming that the Mov-7 and Mov-9 loci represent different integration sites of M-MuLV. Both clones induced XC plaques in a transfection assay. The specific infectivity of the clones, however, was different. A total of 10(-5) XC plaques per genome equivalent were induced by the Mov-9 clone, whereas only 10(-9) XC plaques per genome equivalent were induced by the Mov-7 clone. Moreover, NIH 3T3 cells transfected with the Mov-9 clone produced NB-tropic M-MuLV, whereas cells transfected with the Mov-7 clone did not produce infectious virus. The results suggest that M-MuLV integrated at the Mov-7 locus carries a mutation which prevents synthesis of infectious virus but permits XC plaque induction by partial genome expression or synthesis of noninfectious particles. Thus, the pattern of virus expression in Mov-7 and Mov-9 mice correlates with the biological properties of the respective clones. Genomic DNA from Mov-9 mice was not infectious in the transfection assay (specific infectivity < 10(-7) PFU per genome equivalent). As the only difference between the genomic and the cloned Mov-9 DNA appears to be the presence of 5-methylcytosine in CpG sequences, our results suggest that removal of methyl groups by molecular cloning in procaryotes permits genome expression in transfected eucaryotic cells. Our results support the hypothesis that DNA methylation is relevant not only in genome expression in the animal but also in expression of genes transfected into eucaryotic cells.

Experimental insertion of a retrovirus into the germ line of mice has resulted in an embryonic recessive lethal mutation. Integration of the proviral genome occurred at the 5' end of the alpha 1(I) collagen gene, leading to complete transcriptional block. Developmental arrest of embryos homozygous at the mutated allele coincides with high expression of the gene in normal embryogenesis. Insertion mutagenesis by retroviruses may offer a general approach to the identification and isolation of genes which are transcriptionally active during mammalian development.

The chromatin structure of the collagen alpha 1(I) gene, which has been mutated by retrovirus insertion in Mov13 mice, was compared with that of the wildtype allele. Limited digestions with DNAase I revealed the presence of two hypersensitive sites in all normal cells analyzed, while a third site at 100 to 200 bp 5' of the transcription start was detected only in cells synthesizing collagen alpha 1(I) mRNA. This transcription-associated site was not present in chromatin of the mutant allele, while the two other hypersensitive sites, one of which is located close to the provirus, were not changed by the virus integration. Our results suggest that the virus insertion in Mov13 mice may prevent the developmentally regulated appearance of a transcription-associated hypersensitive site, thereby interfering with proper activation of the gene during embryonic development.