Targeted disruption of the murine dihydrolipoamide dehydrogenase gene (Dld) results in perigastrulation lethality

The Dld gene product, known as dihydrolipoamide dehydrogenase or the E3 component, catalyzes the oxidation of dihydrolipoyl moieties of four mitochondrial multienzyme complexes: pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched-chain α-ketoacid dehydrogenase, and the glycine cleavage system. Deficiency of E3 activity in humans results in various degrees of neurological dysfunction and organic acidosis caused by accumulation of branched-chain amino acids and lactic acid. In this study, we have introduced a null mutation into the murine Dld gene (Dldtm1mjp). The heterozygous animals are shown to have approximately half of wild-type activity levels for E3 and all affected multienzyme complexes but are phenotypically normal. In contrast, the Dld−/− class dies prenatally with apparent developmental delay at 7.5 days postcoitum followed by resorption by 9.5 days postcoitum. The Dld−/− embryos cease to develop at a time shortly after implantation into the uterine wall when most of the embryos have begun to gastrulate. This null phenotype provides in vivo evidence for the requirement of a mitochondrial oxidative pathway during the perigastrulation period. Furthermore, the early prenatal lethal condition of the complete deficiency state may explain the low incidence of detectable cases of E3 deficiency in humans.

A very limited number of keywords (main patterns) describes all sequences of the human variable heavy (VH) and κ (Vκ) domains

Sequences of the variable heavy (VH) and κ (Vκ) domains of Ig structures were divided into 21 fragments that correspond to strands, loops, or parts of these structural units of the variable domains. Amino acid sequences of fragments (termed “words”) were collected from the 1,172 human heavy and 668 human κ chains available in the Kabat database. Statistical analysis of words of 17 fragments was performed (fragments that comprise the complementary determining regions′ fragments will not be discussed in this paper). The number of different words (those with different residues in at least one position) ranged, for various fragments, from 11 to 75 in the κ chains, and from 23 to 189 in the heavy chains. The main result of this study is that very few keywords, or main patterns of words, were necessary to describe over 90% of the sequences (no more than two keywords per fragment in the κ and no more than five per fragment in the heavy chains). No identical keywords were found for different fragments of the variable domains. Keywords of aligned fragments of the VH and Vκ domains were different in all but two instances. Thus, knowing the keywords, one can determine whether any given small part of a sequence belongs to a heavy or κ chain and predict its precise localization in the sequence. In addition, by using all of the keywords obtained through analysis of the Kabat database, it was possible to describe completely the sequences of the human VH and Vκ germ-line segments.

A multimeric complex and the nuclear targeting of the Drosophila Rel protein Dorsal

The intracellular part of the Rel signal transduction pathway in Drosophila is encoded by Toll, tube, pelle, dorsal, and cactus, and it functions to form the dorsal–ventral axis in the Drosophila embryo. Upon activation of the transmembrane receptor Toll, Dorsal dissociates from its cytoplasmic inhibitor Cactus and enters the nucleus. Tube and Pelle are required to relay the signal from Toll to the Dorsal–Cactus complex. In a yeast two-hybrid assay, we found that both Tube and Pelle interact with Dorsal. We confirmed these interactions in an in vitro binding assay. Tube interacts with Dorsal via its C-terminal domain, whereas full-length Pelle is required for Dorsal binding. Tube and Pelle bind Dorsal in the N-terminal domain 1 of the Dorsal Rel homology region rather than at the Cactus binding site. Domain 1 has been found to be necessary for Dorsal nuclear targeting. Genetic experiments indicate that Tube–Dorsal interaction is necessary for normal signal transduction. We propose a model in which Tube, Pelle, Cactus, and Dorsal form a multimeric complex that represents an essential aspect of signal transduction.

Evolutionary instability of the major histocompatibility complex class I loci in New World primates

Homologues of the human major histocompatibility complex (MHC) HLA-A, -B, -E, -F, and -G loci are present in all the Catarrhini (Old World primates, apes, and humans), and some of their allelic lineages have survived several speciation events. Analysis of 26 MHC class I cDNAs from seven different genera of New World primates revealed that the Callitrichinae (tamarins and marmosets) are an exception to these rules of MHC stability. In gene trees of primate MHC class I genes, sequences from the Callitrichinae cluster in a genus-specific fashion, whereas in the other genera of New World primates, as in the Catarrhini, they cluster in a transgeneric way. The genus-specific clustering of the Callitrichinae cDNAs indicates that there is no orthology between MHC class I loci in genera of this phyletic group. Additionally, the Callitrichinae genera exhibit limited variability of their MHC class I genes, in contrast to the high variability displayed by all other primates. Each Callitrichinae genus, therefore, expresses its own set of MHC class I genes, suggesting that an unusually high rate of turnover of loci occurs in this subfamily. The limited variability of MHC class I genes in the Callitrichinae is likely the result of the recent origin of these loci.

In vitro reconstruction of the Aspergillus (= Emericella) nidulans genome

A physical map of the 31-megabase Aspergillus nidulans genome is reported, in which 94% of 5,134 cosmids are assigned to 49 contiguous segments. The physical map is the result of a two-way ordering process, in which clones and probes were ordered simultaneously on a binary DNA/DNA hybridization matrix. Compression by elimination of redundant clones resulted in a minimal map, which is a chromosome walk. Repetitive DNA is nonrandomly dispersed in the A. nidulans genome, reminiscent of heterochromatic banding patterns of higher eukaryotes. We hypothesize gene clusters may arise by horizontal transfer and spread by transposition to explain the nonrandom pattern of repeats along chromosomes.

Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein–Barr virus nuclear antigen 1

The Epstein–Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic T cell epitopes from EBNA4. It appears that the majority of antigens presented via the MHC I pathway are subject to ATP-dependent ubiquitination and degradation by the proteasome. We have investigated the influence of the repeat on this process by comparing the degradation of EBNA1, EBNA4, and Gly-Ala containing EBNA4 chimeras in a cell-free system. EBNA4 was efficiently degraded in an ATP/ubiquitin/proteasome-dependent fashion whereas EBNA1 was resistant to degradation. Processing of EBNA1 was restored by deletion of the Gly-Ala domain whereas insertion of Gly-Ala repeats of various lengths and in different positions prevented the degradation of EBNA4 without appreciable effect on ubiquitination. Inhibition was also achieved by insertion of a Pro-Ala coding sequence. The results suggest that the repeat may affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin/proteasome pathway. The presence of regularly interspersed Ala residues appears to be important for the effect.

Genomic and cDNA sequence analysis of the cell matrix adhesion regulator gene

The cell matrix adhesion regulator (CMAR) gene has been suggested to be a signal transduction molecule influencing cell adhesion to collagen and, through this, possibly involved in tumor suppression. The originally reported CMAR cDNA was 464 bp long with a tyrosine phosphorylation site at the extreme 3′ end, which mutagenesis studies had shown to be central to the function of this gene. Since the discovery of a 4-bp insertion polymorphism within the originally reported coding region, further sequence information has been obtained. The cDNA has been extended 5′ by ≈2 kb revealing a 559-bp region showing strong homology to the proposed 5′ untranslated sequence of a murine protein kinase receptor family member, variant in kinase (vik). CMAR genomic sequencing has shown the presence of an intron, the intron/exon boundary lying within this region of homology. An RNA transcript for CMAR of ≈2.5 kb has also been identified. The data suggest complex mechanisms for control of expression of two closely associated genes, CMAR and the vik- associated sequence.

Sequence of the FRA3B common fragile region: Implications for the mechanism of FHIT deletion

The hypothesis that chromosomal fragile sites may be “weak links” that result in hot spots for cancer-specific chromosome rearrangements was supported by the discovery that numerous cancer cell homozygous deletions and a familial translocation map within the FHIT gene, which encompasses the common fragile site, FRA3B. Sequence analysis of 276 kb of the FRA3B/FHIT locus and 22 associated cancer cell deletion endpoints shows that this locus is a frequent target of homologous recombination between long interspersed nuclear element sequences resulting in FHIT gene internal deletions, probably as a result of carcinogen-induced damage at FRA3B fragile sites.

Comparative mapping of the human 22q11 chromosomal region and the orthologous region in mice reveals complex changes in gene organization

The region of human chromosome 22q11 is prone to rearrangements. The resulting chromosomal abnormalities are involved in Velo-cardio-facial and DiGeorge syndromes (VCFS and DGS) (deletions), “cat eye” syndrome (duplications), and certain types of tumors (translocations). As a prelude to the development of mouse models for VCFS/DGS by generating targeted deletions in the mouse genome, we examined the organization of genes from human chromosome 22q11 in the mouse. Using genetic linkage analysis and detailed physical mapping, we show that genes from a relatively small region of human 22q11 are distributed on three mouse chromosomes (MMU6, MMU10, and MMU16). Furthermore, although the region corresponding to about 2.5 megabases of the VCFS/DGS critical region is located on mouse chromosome 16, the relative organization of the region is quite different from that in humans. Our results show that the instability of the 22q11 region is not restricted to humans but may have been present throughout evolution. The results also underscore the importance of detailed comparative mapping of genes in mice and humans as a prerequisite for the development of mouse models of human diseases involving chromosomal rearrangements.

Role of B cells in antigen presentation of the hepatitis B core

The hepatitis B virus (HBV) nucleocapsid or core antigen (HBcAg) is extremely immunogenic during infection and after immunization. For example, during many chronic infections, HBcAg is the only antigen capable of eliciting an immune response, and nanogram amounts of HBcAg elicit antibody production in mice. Recent structural analysis has revealed a number of characteristics that may help explain this potent immunogenicity. Our analysis of how the HBcAg is presented to the immune system revealed that the HBcAg binds to specific membrane Ig (mIg) antigen receptors on a high frequency of resting, murine B cells sufficiently to induce B7.1 and B7.2 costimulatory molecules. This enables HBcAg-specific B cells from unprimed mice to take up, process, and present HBcAg to naive Th cells in vivo and to T cell hybridomas in vitro approximately 105 times more efficiently than classical macrophage or dendritic antigen-presenting cells (APC). These results reveal a structure–function relation for the HBcAg, confirm that B cells can function as primary APC, explain the enhanced immunogenicity of HBcAg, and may have relevance for the induction and/or maintenance of chronic HBV infection.

Role of the major histocompatibility complex class II Ea gene in lupus susceptibility in mice

The gene(s) encoded within major histocompatibility complex (MHC) act as one of the major genetic elements contributing to the susceptibility of murine systemic lupus erythematosus (SLE). We have recently demonstrated that lupus susceptibility is more closely linked to the I-E− H-2b haplotype than to the I-E+ H-2d haplotype in lupus-prone BXSB and (NZB × BXSB)F1 hybrid mice. To investigate whether the reduced susceptibility to SLE in H-2d mice is related to the expression of the MHC class II Ea gene (absent in H-2b mice), we determined the possible role of the Ea gene as a lupus protective gene in mice. Our results showed that (i) the development of SLE was almost completely prevented in BXSB (H-2b) mice expressing two copies of the Ead transgene at the homozygous level as well as in BXSB H-2k (I-E+) congenic mice as for H-2d BXSB mice, and (ii) the expression of two functional Ea (transgenic and endogenous) genes in either H-2d/b (NZB × BXSB)F1 or H-2k/b (MRL × BXSB)F1 mice provided protection from SLE at levels comparable to those conferred by the H-2d/d or H-2k/k haplotype. In addition, the level of the Ea gene-mediated protection appeared to be dependent on the genetic susceptibility to SLE in individual lupus-prone mice. Our results indicate that the reduced susceptibility associated with the I-E+ H-2d and H-2k haplotypes (versus the I-E− H-2b haplotype) is largely, if not all, contributed by the apparent autoimmune suppressive effect of the Ea gene, independently of the expression of the I-A or other MHC-linked genes.

Regulated expression of the diphtheria toxin A chain by a tumor-specific chimeric transcription factor results in selective toxicity for alveolar rhabdomyosarcoma cells

Alveolar rhabdomyosarcoma (ARMS) cells often harbor one of two unique chromosomal translocations, either t(2;13)(q35;q14) or t(1;13)(p36;q14). The chimeric proteins expressed from these rearrangements, PAX3-FKHR and PAX7-FKHR, respectively, are potent transcriptional activators. In an effort to exploit these unique cancer-specific molecules to achieve ARMS-specific expression of therapeutic genes, we have studied the expression of a minimal promoter linked to six copies of a PAX3 DNA binding site, prs-9. In transient transfections, expression of the prs-9-regulated reporter genes was ≈250-fold higher than expression of genes lacking the prs-9 sequences in cell lines derived from ARMS, but remained at or below baseline levels in other cells. High expression of these prs-9-regulated genes was also observed in a cancer cell line that lacks t(2;13) but was stably transfected with a plasmid expressing PAX3-FKHR. Transfection of a plasmid containing the diphtheria toxin A chain gene regulated by prs-9 sequences (pA3–6PED) was selectively cytotoxic for PAX3-FKHR-expressing cells. This was shown by inhibition of gene expression from cotransfected plasmids and by direct cytotoxicity after transfected cells were isolated by cell sorting. Gene transfer of pA3–6PED may thus be useful as a cancer-specific treatment strategy for t(2;13)- or t(1;13)-positive ARMS. Furthermore, gene transfer of fusion protein-regulated toxin genes might also be applied to the treatment of other cancers that harbor cancer-specific chromosomal translocations involving transcription factors.

p53CP, a putative p53 competing protein that specifically binds to the consensus p53 DNA binding sites: A third member of the p53 family?

p53 tumor suppressor protein negatively regulates cell growth, mainly through the transactivation of its downstream target genes. As a sequence-specific DNA binding transcription factor, p53 specifically binds to a 20-bp consensus motif 5′-PuPuPuC(A/T) (T/A)GPyPyPyPuPuPuC(A/T)(T/A)GPyPyPy-3′. We have now identified, partially purified, and characterized an additional ≈40-kDa nuclear protein, p53CP (p53 competing protein), that specifically binds to the consensus p53 binding sites found in several p53 downstream target genes, including Waf-1, Gadd45, Mdm2, Bax, and RGC. The minimal sequence requirement for binding is a 14-bp motif, 5′-CTTGCTTGAACAGG-3′ [5′-C(A/T)(T/A)GPyPyPyPuPuPuC(A/T)(T/A)G-3′], which includes the central nucleotides of the typical p53 binding site with one mismatch. p53CP and p53 (complexed with antibody) showed a similar binding specificity to Waf-1 site but differences in Gadd45 and T3SF binding. Like p53, p53CP also binds both double- and single-stranded DNA oligonucleotides. Important to note, cell cycle blockers and DNA damaging reagents, which induce p53 binding activity, were found to inhibit p53CP binding in p53-positive, but not in p53-negative, cells. This finding suggested a p53-dependent coordinate regulation of p53 and p53CP in response to external stimuli. p53CP therefore could be a third member of the p53 family, in addition to p53 and p73, a newly identified p53 homolog. p53CP, if sequestering p53 from its DNA binding sites through competitive binding, may provide a novel mechanism of p53 inactivation. Alternatively, p53CP may have p53-like functions by binding and transactivating p53 downstream target genes. Cloning of the p53CP gene ultimately will resolve this issue.

Targeted deletion of all isoforms of the trkC gene suggests the use of alternate receptors by its ligand neurotrophin-3 in neuronal development and implicates trkC in normal cardiogenesis

We have generated null mutant mice that lack expression of all isoforms encoded by the trkC locus. These mice display a behavioral phenotype characterized by a loss of proprioceptive neurons. Neuronal counts of sensory ganglia in the trkC mutant mice reveal less severe losses than those in NT-3 null mutant mice, strongly suggesting that NT-3, in vivo, may signal through receptors other than trkC. Mice lacking either NT-3 or all trkC receptor isoforms die in the early postnatal period. Histological examination of trkC-deficient mice reveals severe cardiac defects such as atrial and ventricular septal defects, and valvular defects including pulmonic stenosis. Formation of these structures during development is dependent on cardiac neural crest function. The similarities in cardiac defects observed in the trkC and NT-3 null mutant mice indicate that the trkC receptor mediates most NT-3 effects on the cardiac neural crest.

Ca2+-dependent and -independent interactions of the isoforms of the α1A subunit of brain Ca2+ channels with presynaptic SNARE proteins

Fast neurotransmission requires that docked synaptic vesicles be located near the presynaptic N-type or P/Q-type calcium channels. Specific protein–protein interactions between a synaptic protein interaction (synprint) site on N-type and P/Q-type channels and the presynaptic SNARE proteins syntaxin, SNAP-25, and synaptotagmin are required for efficient, synchronous neurotransmitter release. Interaction of the synprint site of N-type calcium channels with syntaxin and SNAP-25 has a biphasic calcium dependence with maximal binding at 10–20 μM. We report here that the synprint sites of the BI and rbA isoforms of the α1A subunit of P/Q-type Ca2+ channels have different patterns of interactions with synaptic proteins. The BI isoform of α1A specifically interacts with syntaxin, SNAP-25, and synaptotagmin independent of Ca2+ concentration and binds with high affinity to the C2B domain of synaptotagmin but not the C2A domain. The rbA isoform of α1A interacts specifically with synaptotagmin and SNAP-25 but not with syntaxin. Binding of synaptotagmin to the rbA isoform of α1A is Ca2+-dependent, with maximum affinity at 10–20 μM Ca2+. Although the rbA isoform of α1A binds well to both the C2A and C2B domains of synaptotagmin, only the interaction with the C2A domain is Ca2+-dependent. These differential, Ca2+-dependent interactions of Ca2+ channel synprint sites with SNARE proteins may modulate the efficiency of transmitter release triggered by Ca2+ influx through these channels.