Pip92: a short-lived, growth factor-inducible protein in BALB/c 3T3 and PC12 cells.

pip92 is a cellular immediate-early gene inducible by serum growth factors in fibroblasts. It is also induced in the rat pheochromocytoma cell line PC12 by agents that cause proliferation, neuronal differentiation, and membrane depolarization. We show that the pip92-encoded polypeptide is a proline-rich protein of 221 amino acids, has an extremely short half-life, and is localized in the cytoplasm. We hypothesize that Pip92 plays a role in mediating the cellular responses to a variety of extracellular signals.     

p-Aminobenzoate-p-aminobenzoate.

p-Aminobenzoate (PABA) synthase from Bacillus subtilis is an aggregate composed of two nonidentical subunits and has the following properties. (i) In crude extracts this enzyme catalyzes the formation of PABA in the presence of chorismate and either glutamine (amidotransferase) or ammonia (aminase). The amidotransferase activity is about 5- to 10-fold higher than the aminase activity and is stable for at least 1 week when frozen at -70 C. (II) Although no divalent cation requirement could be demonstrated with crude extracts, 2 mM ethylene-diaminetetraacetic acid completely inhibits both activities. (iii) After ammonium sulfate fractionation both the aminase and amidotransferase activities require Mg2+ and guanosine in addition to the substrates indicated above for optimal activity. The guanosine requirement can be replaced by guanosine 5'-monophosphate, guanosine 5'-diphosphate, and guanosine 5'-triphosphate but not by guanine, adenosine 5'-triphosphate, uridine 5'-triphosphate, cytidine 5'-triphosphate, thymidine 5'-triphosphate, inorganic phosphate, and phosphoribosylpyrophosphate. Furthermore, at a pH above 7.4 or below 6.4 activity is rapidly lost a 4 C, or -60 C. (IV) The enzyme is composed of two non-identical subunits, designated subunit A and subunit X. Subunit A has an estimated molecular weight of 31,000, whereas subunit X has an estimated molecular weight of 19,000. Subunit A has aminase activity but no amidotransferase activity; a mutation at the pabA locus results in the loss of PABA synthase activity. Subunit X, which is also a component of the anthranilate synthase complex, has no PABA synthase activity itself but complexes with subunit A to give an AX aggregate that can use glutamine as a substrate. (v) The molecular weight of the AX complex has been estimated at 50,000, suggesting a 1:1 ratio of subunits. (vi) The enzyme is readily associated and dissociated.     

Linkage of DNA markers to cystic fibrosis in 26 families.

Two DNA markers, the met oncogene and the anonymous probe, pJ3.11, previously reported to be tightly linked to cystic fibrosis (CF), were used for linkage analysis in 26 families with two or more individuals affected with CF. A new high frequency polymorphism was identified using BanI and the pmetD probe. The results of linkage analysis were as follows: between met and CF, lod score of 18.2 at theta of .009; between pJ3.11 and CF, lod score of 12.1 at theta of 0; and between met and pJ3.11, lod score of 16.7 at theta of 0. These data indicate that most or all of CF is due to an abnormality at a single locus and that the DNA markers are useful for prenatal diagnosis and heterozygote detection within affected families.     

Identification of human immunodeficiency virus envelope gene sequences influencing viral entry into CD4-positive HeLa cells, T-leukemia cells, and macrophages.

Infectious recombinant viruses were constructed from three molecularly cloned human immunodeficiency virus (HIV) strains varying in cell tropism. All recombinants showed a high infectivity titer on phytohemagglutinin-stimulated normal T lymphocytes. However, a 120-bp region of the envelope gene including the area of the V3 hypervariable loop was found to influence infectivity titer on both clone 1022 CD4-positive HeLa cells and CD4-positive CEM leukemia cells. Infectivity for macrophages was more complex. All viruses replicated in macrophages to a low level, but viral sequences both inside and outside the V3 loop region influenced the efficiency of replication. Two experiments showed that the mechanism of restriction of infection of 1022 cells by HIV strain JR-CSF was related to lack of virus entry. First, productive virus infection occurred after transfection of 1022 cells with viral plasmid DNA. Second, the nonpermissive HIV strain JR-CSF could infect 1022 cells when pseudotyped with the envelope of other retroviruses, including human T-cell leukemia virus type I (HTLV-I), HTLV-II, and amphotropic murine leukemia virus. These results demonstrate the possibility that unexpected cell types might be infected with HIV in human patients coinfected with HIV and HTLV-I or HTLV-II.     

Identification of three amino acid residues in the B subunit of Shiga toxin and Shiga-like toxin type II that are essential for holotoxin activity.

Shiga toxin of Shigella dysenteriae type I and Shiga-like toxins I and II (SLT-I and SLT-II, respectively) of enterohemorrhagic Escherichia coli are functionally similar protein cytotoxins. These toxin molecules have a bipartite molecular structure which consists of an enzymatically active A subunit that inhibits protein synthesis in eukaryotic cells and an oligomeric B subunit that binds to globotriaosylceramide glycolipid receptors on eukaryotic cells. Regionally directed chemical mutagenesis of the B subunit of SLT-II was used to identify amino acids in the B subunit that are critical for SLT-II holotoxin cytotoxic activity. Three noncytotoxic mutants were isolated, and their mutations were mapped. The substitutions of arginine with cysteine at codon 32, alanine with threonine at codon 42, and glycine with aspartic acid at codon 59 in the 70-amino-acid mature SLT-II B polypeptide resulted in the complete abolition of cytotoxicity. The analogous arginine, alanine, and glycine residues were conserved at codons 33, 43, and 60 in the 69-amino-acid mature B polypeptide of Shiga toxin. Comparable mutations induced in the B-subunit gene of Shiga toxin by oligonucleotide-directed, site-specific mutagenesis resulted in drastically decreased cytotoxicity (10(3)- to 10(6)-fold) as compared with that of wild-type Shiga toxin. The mutant SLT-II and Shiga toxin B subunits were characterized for stability, receptor binding, immunoreactivity, and ability to be assembled into holotoxin.     

Genetic mapping of the beta 1 GABA receptor gene to human chromosome 4, using a tetranucleotide repeat polymorphism.

As more coding loci for functional human genes are described, there is a growing need to identify DNA polymorphisms in specific genes. By examining DNA sequences within the introns of the beta 1 subunit of the gamma-aminobutyric acid receptor gene, GABARB1, we found a tetranucleotide repeat sequence (GATA). Amplification of this region by using PCR revealed seven alleles and a high degree of polymorphism (PIC = .75) in human populations. DNAs from the CEPH families were typed for the GABARB1 intron polymorphism and were analyzed with respect to 20 linked markers on chromosome 4. The results permit placement of GABARB1 on the linkage map of chromosome 4, between D4S104 and ALB. These results affirm that sequence analysis of noncoding segments included within or adjacent to functional genes has value as a strategy to detect highly informative polymorphisms.     

The human myelin-basic-protein gene: chromosomal localization and RFLP analysis.

With a human myelin-basic-protein (MBP) cDNA used as a probe, the human MBP gene has been mapped to chromosome region 18q22-q23 by a combination of Southern hybridization to a panel of somatic-cell hybrid DNAs and in situ hybridization to metaphase chromosomes. Restriction-fragment-length polymorphisms (RFLPs) have also been identified with this probe in human DNA, by means of the restriction enzymes BamHI, PvuII, and PstI. In studies of informative families, the alleles of the BamHI and PvuII polymorphisms have been shown to segregate as Mendelian traits.     

Cloning and sequencing of a Shiga-like toxin type II variant from Escherichia coli strain responsible for edema disease of swine.

A Shiga-like toxin type II variant (SLT-IIv) is produced by strains of Escherichia coli responsible for edema disease of swine and is antigenically related to Shiga-like toxin type II (SLT-II) of enterohemorrhagic E. coli. However, SLT-IIv is only active against Vero cells, whereas SLT-II is active against both Vero and HeLa cells. The structural genes for SLT-IIv were cloned from E. coli S1191, and the nucleotide sequence was determined and compared with those of other members of the Shiga toxin family. The A subunit genes for SLT-IIv and SLT-II were highly homologous (94%), whereas the B subunit genes were less homologous (79%). The SLT-IIv genes were more distantly related (55 to 60% overall homology) to the genes for Shiga toxin of Shigella dysenteriae type 1 and the nearly identical Shiga-like toxin type I (SLT-I) of enterohemorrhagic E. coli. (These toxins are referred to together as Shiga toxin/SLT-I.) The A subunit of SLT-IIv, like those of other members of this toxin family, had regions of homology with the plant lectin ricin. SLT-IIv did not bind to galactose-alpha 1-4-galactose conjugated to bovine serum albumin, which is an analog of the eucaryotic cell receptor for Shiga toxin/SLT-I and SLT-II. These findings support the hypothesis that SLT-IIv binds to a different cellular receptor than do other members of the Shiga toxin family but has a similar mode of intracellular action. The organization of the SLT-IIv operon was similar to that of other members of the Shiga toxin family. Iron did not suppress SLT-IIv or SLT-II production, in contrast with its effect on Shiga toxin/SLT-I. Therefore, the regulation of synthesis of SLT-IIv and SLT-II differs from that of Shiga toxin/SLT-I.