The cDNA sequence and chromosomal location of the murine GABAA alpha 1 receptor gene.

The murine GABAA/benzodiazepine (GABAA/BZ) receptor alpha 1 subunit cDNA has been isolated from a BALB/c mouse brain library and sequenced. The cDNA is 2665 nucleotides long with an open reading frame of 455 amino acids. It shows significant homology to the GABAA receptor alpha 1 subunit cDNA sequences of other species. Excluding deletions, the murine GABAA alpha 1 receptor exhibits 96% nucleotide and 100% amino acid sequence homology to the rat alpha 1 receptor cDNA and over 91% nucleotide and 98% amino acid sequence homology to the bovine and human alpha 1 receptor cDNAs in the protein coding region. This murine cDNA was used to locate the alpha 1 receptor subunit gene, Gabra-1, to murine Chromosome 11 between Il-3 and Rel. This assignment extends proximally the segment of mouse Chromosome 11 with known homology to human chromosome 5.     

LRP5 and LRP6 are not required for protective antigen-mediated internalization or lethality of anthrax lethal toxin

Anthrax toxin (AnTx) plays a key role in the pathogenesis of anthrax. AnTx is composed of three proteins: protective antigen (PA), edema factor, and lethal factor (LF). PA is not toxic but serves to bind cells and translocate the toxic edema factor or LF moieties to the cytosol. Recently, the low-density lipoprotein receptor-related protein LRP6 has been reported to mediate internalization and lethality of AnTx. Based on its similarity to LRP6, we hypothesized that LRP5 may also play a role in cellular uptake of AnTx. We assayed PA-dependent uptake of anthrax LF or a cytotoxic LF fusion protein (FP59) in cells and mice harboring targeted deletions of Lrp5 or Lrp6. Unexpectedly, we observed that uptake was unaltered in the presence or absence of either Lrp5 or Lrp6 expression. Moreover, we observed efficient PA-mediated uptake into anthrax toxin receptor (ANTXR)-deficient Chinese hamster ovary cells (PR230) that had been stably engineered to express either human ANTXR1 or human ANTXR2 in the presence or absence of siRNA specific for LRP5 or LRP6. Our results demonstrate that neither LRP5 nor LRP6 is necessary for PA-mediated internalization or lethality of anthrax lethal toxin.     

一个新的东亚钳蝎毒素(BmKT_1)全长cDNA的克隆和分析

首先构建了东亚钳蝎毒腺组织 c DNA文库 ;根据已知的东亚钳蝎哺乳动物毒素氨基酸序列保守区设计引物 ,并用 PCR从 c DNA文库中扩增出一个 c DNA片段作为筛选 c DNA文库的探针 ;从 c DNA文库中筛选到二个编码同一个新的蝎毒素多肽的 c DNA,它们除 3′- UTR外 ,其余序列完全一致 .它们均含有 2 55bp长的开放阅读框 ,编码 85肽的前体毒素 ,包括 1 9个氨基酸残基的信号肽 ,66个残基的成熟毒素 (命名为 Bm KT1) ;Bm KT1氨基酸序列与已知的蝎毒素具有较大的同源性 ,与 Bm KM1,Lqq ,Lqhα IT和 Bm K M10 的同源性分别为 77%、67%、67%和 65% .Bm KT1的 C端不存在末端修饰步骤且具有一个与这些毒素不相同的特征结构 ,即在末端延伸了两个氨基酸残基 - P- S,推测 Bm KT1具有新的活性功能特征 .     

Reciprocal regulation of sex-dependent expression of testosterone 15 alpha-hydroxylase (P-450(15 alpha)) in liver and kidney of male mice by androgen. Evidence for a single gene

Testosterone 15 alpha-hydroxylase activities and its mRNA levels are higher in kidneys than in livers from male 129/J mice. Castration of 129/J male mice resulted in repression of P-450(15 alpha) in kidney, but increased it in liver. Two types of cDNA (p15 alpha-29 (Type I) and -15 (Type II)) encoding P-450(15 alpha) were previously cloned from 129/J female livers (Burkhart, B.A., Harada, N., and Negishi, M. (1985) J. Biol. Chem. 260, 15357-15361). With the use of p15 alpha-29 as a probe, Type I and II P-450(15 alpha) cDNAs were isolated from libraries of 129/J kidney poly(A)+ RNA. The nucleotide sequences of the cDNAs showed that Type I and II cDNAs from liver and kidney were identical and shared 98.3% similarity. The deduced amino acid sequence from a full-length Type I cDNA indicated that Type I P-450(15 alpha) consists of 494 amino acids with a molecular weight of 56,594. Nine amino acid substitutions were found in the Type II clone in 432 amino acids overlapping Type I. Type I cDNA clones accounted for approximately 90% P-450(15 alpha) clones isolated from a male kidney library, whereas approximately 90% of cDNA clones in a female kidney library were Type II. Liver cDNA libraries from males and females contained similar ratios of Type I and II. Effects of castration on Type I and II mRNAs were determined by Southern hybridization of a 32P-labeled ClaI-ClaI fragment from p15 alpha-29 to cDNAs synthesized from kidney and liver poly(A)+ RNAs prepared from sham-operated, castrated 129/J mice. The double-stranded cDNAs were digested with ClaI and PstI prior to gel electrophoresis to create the diagnostic restriction fragments specific for Type I or II. Castration resulted in decreased levels of Type I mRNA in male kidney. In male liver, only Type I mRNA rose significantly in response to castration. Testosterone administration returned the Type I mRNA to normal levels in castrated mice. It therefore appears that the high levels of P-450(15 alpha) in male kidney were due to androgen-dependent induction of Type I mRNA. Both Types I and II were repressed in male liver, which results in decreased levels of P-450(15 alpha). Androgen was responsible for the repression and expression of Type I in liver and kidney, but not Type II.     

Restriction analysis of cDNA for cow alpha s1-, beta- and kappa-caseins

By means of in situ hybridization to cloned cDNA fragments coding for cow alpha s1-, beta- and kappa-caseins, screening of the library of clones containing the cDNA complementary to mRNA of lactating cow mammary gland was carried out. The clones containing the sequences of alpha s1-, beta- and kappa-casein cDNAs were shown to constitute 4.0, 3.2 and 0.7% of all the colonies, respectively. The analysis of the data on cross-hybridization points to the absence of extensive regions of homology between the above-mentioned cDNAs. The restriction analysis of cDNAs of the selected clones was carried out and the restriction maps of cDNAs of these three caseins were constructed. The restriction analysis data and determination of the nucleotide sequence of 5'-termini of the studied cDNAs indicated that the cloned sequences were the full-length mRNA copies of alpha s1-, beta- and kappa-caseins. The data obtained on restriction analysis are utilized in mapping the corresponding natural genes of cow caseins.     

37-kDa laminin receptor precursor modulates cytotoxic necrotizing factor 1-mediated RhoA activation and bacterial uptake

Cytotoxic necrotizing factor 1 (CNF1) is a bacterial toxin known to activate Rho GTPases and induce host cell cytoskeleton rearrangements. The constitutive activation of Rho GTPases by CNF1 is shown to enhance bacterial uptake in epithelial cells and human brain microvascular endothelial cells. However, it is unknown how exogenous CNF1 exhibits such phenotypes in eukaryotic cells. Here, we identified 37-kDa laminin receptor precursor (LRP) as the receptor for CNF1 from screening the cDNA library of human brain microvascular endothelial cells by the yeast two-hybrid system using the N-terminal domain of CNF1 as bait. CNF1-mediated RhoA activation and bacterial uptake were inhibited by exogenous LRP or LRP antisense oligodeoxynucleotides, whereas they were increased in LRP-overexpressing cells. These findings indicate that the CNF1 interaction with LRP is the initial step required for CNF1-mediated RhoA activation and bacterial uptake in eukaryotic cells.     

Human apolipoprotein E mRNA. cDNA cloning and nucleotide sequencing of a new variant

The complete nucleotide sequences of three cloned cDNAs corresponding to human liver apolipoprotein E (apo-E) mRNA were determined. Analysis of the longest cDNA showed that it contained 1157 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 61 nucleotides, a signal peptide region corresponding to 18 amino acids, a mature protein region corresponding to 299 amino acids, and a 3'-terminal nontranslated region of 142 nucleotides. The inferred amino acid sequences from two cDNAs were identical and corresponded to the amino acid sequence for plasma apo-E3 that has been reported previously ( Rall , S. C., Jr., Weisgraber , K. H., and Mahley , R. W. (1982) J. Biol. Chem. 257, 4171-4178). The third cDNA differed from the other two cDNAs in five nucleotide positions. Three of these differences occurred in the third nucleotide position of amino acid codons, resulting in no change in the corresponding amino acids at residues Val-85, Ser-223, and Gln-248. The other two altered nucleotides occurred in the first nucleotide position of codons, leading to changes in the amino acids encoded. In the variant sequence, a threonine replaced the normal alanine at residue 99 and a proline replaced the normal alanine at residue 152. We have concluded that the human liver donor was heterozygous for the epsilon 3 genotype. The variant cDNA corresponds to a new, previously undescribed variant form of apo-E in which the amino acid substitutions of the protein are electrophoretically silent; it would probably be undetectable by standard apo-E phenotyping methods. The amino acid substitution at position 152 occurs in a region of apo-E that appears to be important for receptor binding, and it may have clinical significance.     

Phanerochaete chrysosporium glyoxal oxidase is encoded by two allelic variants: structure, genomic organization, and heterologous expression of glx1 and glx2.

A cDNA clone (glx-2c) encoding glyoxal oxidase (GLOX) was isolated from a Phanerochaete chrysosporium lambda gt11 library, and its nucleotide sequence was shown to be distinct from that of the previously described clone glx-1c (P. J. Kersten and D. Cullen, Proc. Natl. Acad. Sci. USA 90:7411-7413, 1993). Genomic clones corresponding to both cDNAs were also isolated and sequenced. overall nucleotide sequence identity was 98%, and the predicted proteins differed by a single residue: Lys-308<==>Thr-308. Analyses of parental dikaryotic strain BKM-F-1767 and homokaryotic progeny firmly established allelism for these structural variants. Southern blots of pulsed-field gels localized the GLOX gene (glx) to a dimorphic chromosome separate from the peroxidase and cellobiohydrolase genes of P. chrysosporium. Controlled expression of active GLOX was obtained from Aspergillus nidulans transformants when glx-1c was fused to the promoter and secretion signal of the A. niger glucoamylase gene. The GLOX isozyme corresponding to glx-2c was also efficiently secreted by A. nidulans following site-specific mutagenesis of the expression vector at codon 308 of glx-1c.     

Mutant Lrp1 knock-in mice generated by recombinase-mediated cassette exchange reveal differential importance of the NPXY motifs in the intracellular domain of LRP1 for normal fetal development

Lrp1 knock-in mice carrying either a wild-type allele or three different mutated alleles encoding the multifunctional endocytic receptor LRP1 were generated by recombinase-mediated cassette exchange (RMCE). Reinsertion by RMCE of a wild-type allele led to a normal pattern and level of gene expression and a completely normal phenotype, indicating that the RMCE procedure itself is neutral with respect to the function of the gene locus. In contrast, reinsertion of mutated LRP1 alleles carrying either inactivating mutations in the proximal NPXY motif (NPTY-->AATA) of the cytoplasmic domain or in the furin cleavage site (RHRR-->AHAA) caused distinctive liver phenotypes: respectively, either a late fetal destruction of the organ causing perinatal death or a selective enlargement of von-Kupffer cell lysosomes reminiscent of a mild lysosomal storage without an apparent negative effect on animal survival. Notably, mutation of the distal NPXY motif overlapping with an YXXL motif (NPVYATL-->AAVAATL) did not cause any obvious pathological effect. The mutations showed no effect on the LRP1 expression level; however, as expected, the proteolytic maturation of LRP1 into its two subunits was significantly impaired, although not completely abolished, in the furin cleavage mutant. These data demonstrate that RMCE is a reliable and efficient approach to generate multiple mutant knock-in alleles for in vivo functional analysis of individual domains or motifs of large multidomain proteins. Its application in Lrp1 reveals dramatically variant phenotypes, of which further characterization will definitively contribute to our understanding of the biology of this multifunctional receptor.     

Mammalian cell expression of an active site mutant of Pseudomonas exotoxin disrupts LRP1 maturation

Low density lipoprotein receptor-related protein 1, (LRP1) is a large multifunctional receptor that binds more than 25 physiologic ligands. In addition, it functions as the surface receptor for several Rhinoviruses, HIV-tat and Pseudomonas exotoxin (PE). We report that the expression of PE within mammalian cells can serve as a probe of LRP1 maturation and functionality. To avoid cell killing, an enzymatically inactive form of the toxin (PEΔ553) was expressed. A permanent cell line (termed CY301) was established whereby PEΔ553 was expressed continually into the ER of CHO cells. CY301 cells were 100-fold resistant to exogenously added active PE but exhibited no cross-resistance to other toxins. Our studies indicate that PEΔ553 bound to immature LRP1 in the ER, prevented its maturation to the cell surface and thereby produced a toxin resistant phenotype. By confocal microscopy, cell-associated PEΔ553 was localized to the ER and co-localized with LRP1. Further characterization of CY301 cells indicated that RAP, the chaperone that aids in LRP1 folding, was released to the growth media. Thus the intracellular expression of PEΔ553 appears to be a valuable probe of LRP1 maturation and trafficking.     

Lipoprotein receptor LRP1 regulates leptin signaling and energy homeostasis in the adult central nervous system

Obesity is a growing epidemic characterized by excess fat storage in adipocytes. Although lipoprotein receptors play important roles in lipid uptake, their role in controlling food intake and obesity is not known. Here we show that the lipoprotein receptor LRP1 regulates leptin signaling and energy homeostasis. Conditional deletion of the Lrp1 gene in the brain resulted in an obese phenotype characterized by increased food intake, decreased energy consumption, and decreased leptin signaling. LRP1 directly binds to leptin and the leptin receptor complex and is required for leptin receptor phosphorylation and Stat3 activation. We further showed that deletion of the Lrp1 gene specifically in the hypothalamus by Cre lentivirus injection is sufficient to trigger accelerated weight gain. Together, our results demonstrate that the lipoprotein receptor LRP1, which is critical in lipid metabolism, also regulates food intake and energy homeostasis in the adult central nervous system.     

Truncated forms of the dual function human ASCT2 neutral amino acid transporter/retroviral receptor are translationally initiated at multiple alternative CUG and GUG codons

The sodium-dependent neutral amino acid transporter type 2 (ASCT2) was recently identified as a cell surface receptor for endogenously inherited retroviruses of cats, baboons, and humans as well as for horizontally transmitted type-D simian retroviruses. By functional cloning, we obtained 10 full-length 2.9-kilobase pair (kbp) cDNAs and two smaller identical 2.1-kbp cDNAs that conferred susceptibility to these viruses. Compared with the 2.9-kbp cDNA, the 2.1-kbp cDNA contains exonic deletions in its 3' noncoding region and a 627-bp 5' truncation that eliminates sequences encoding the amino-terminal portion of the full-length ASCT2 protein. Although expression of the truncated mRNA caused enhanced amino acid transport and viral receptor activities, the AUG codon nearest to its 5' end is flanked by nucleotides that are incompatible with translational initiation and the next in-frame AUG codon is far downstream toward the end of the protein coding sequence. Interestingly, the 5' region of the truncated ASCT2 mRNA contains a closely linked series of CUG(Leu) and GUG(Val) codons in optimal consensus contexts for translational initiation. By deletion and site-directed mutagenesis, cell-free translation, and analyses of epitope-tagged ASCT2 proteins synthesized intracellularly, we determined that the truncated mRNA encodes multiple ASCT2 isoforms with distinct amino termini that are translationally initiated by a leaky scanning mechanism at these CUG and GUG codons. Although the full-length ASCT2 mRNA contains a 5'-situated AUG initiation codon, a significant degree of leaky scanning also occurred in its translation. ASCT2 isoforms with relatively short truncations were active in both amino acid transport and viral reception, whereas an isoform with a 79-amino acid truncation that lacked the first transmembrane sequence was active only in viral reception. We conclude that ASCT2 isoforms with truncated amino termini are synthesized in mammalian cells by a leaky scanning mechanism that employs multiple alternative CUG and GUG initiation codons.     

Defective splicing of Megf7/Lrp4, a regulator of distal limb development, in autosomal recessive mulefoot disease

Mulefoot disease (MFD) is an autosomal recessive disorder of phenotypically variable expression that causes syndactyly in certain strains of cows. MFD maps to a narrow interval on bovine chromosome 15 that is syntenic to human chromosome 11p12-p11.2. This region contains MEGF7/LRP4 (approved gene symbol LRP4), a gene that encodes a member of the multifunctional low-density lipoprotein receptor gene family. Targeted and naturally occurring mutations in the murine Megf7/Lrp4 gene, a putative coreceptor in the Wnt signaling pathway, cause polysyndactyly in the rodent. Thus, Megf7/Lrp4 is a strong candidate for the MFD mutation. Using PCR analysis of tissue samples and sperm from confirmed homozygous MFD carriers, we have identified a functional single base pair mutation in the affected animals. We show that a G --> A transition at the first nucleotide in the splice donor site of intron 37 completely disables this splice site. The abnormal splicing that is caused by this mutation predicts the generation of a dysfunctional membrane-anchored receptor lacking the normal cytoplasmic domain. These findings confirm that autosomal recessive loss-of-function mutations in Megf7/Lrp4 result in phenotypically similar forms of syndactyly in different mammalian species and that such mutations are the cause of MFD in bovines.     

Cloning, sequence analysis, and expression of ligninolytic phenoloxidase genes of the white-rot basidiomycete Coriolus hirsutus

Two cDNAs and two genomic DNAs coding for the allelic forms of the ligninolytic phenoloxidase were isolated from the white-rot fungus Coriolus hirsutus. The cloned genes were identified in genetic libraries by hybridization screening using four deoxyoligonucleotide probes which corresponded to the partial amino acid sequence of the purified enzyme. Each cDNA encoded the full-length of the phenoloxidase, a protein consisting of 499 amino acid residues, and its putative signal peptide of 21 amino acid residues. The nucleotide sequences of the two alleles differed by 18 single base changes within the open reading frames resulting in one amino acid substitution. Ten small introns interrupted both genomic DNAs as indicated by direct comparison with the corresponding cDNAs. Putative eukaryotic regulatory sequences, "CAAT" and "TATA," were observed in the 5'-flanking region of both genomic DNAs. Each of the phenoloxidase cDNAs was successfully expressed in an active form in Saccharomyces cerevisiae using the useful yeast expression vector YEp51.