Identification of novel genes that are differentially expressed in human colorectal carcinoma

To investigate the origin of fragile X mutations in the Argentine population, we studied the alleles and haplotypes at DXS548 and FRAXAC1 loci of 42 unrelated fragile X chromosomes and 168 normal ones. Four haplotypes presented in linkage disequilibrium and accounted for 76.2% of fragile X chromosomes, representing the high frequency of haplotype DXS548-FRAXAC1 7-1 (26.2%) characteristic of our population. FRAXAC1 allele 1 was observed on 47.6% of fragile X chromosomes. Thus, we provide evidence for fragile X founder effects in the Argentine population, similar to those observed in Caucasians and in Asians.     

Major antigenic proteins of the agent of human granulocytic ehrlichiosis are encoded by members of a multigene family

Western blot analysis of proteins from a cell culture isolate (USG3) of the human granulocytic ehrlichiosis (HGE) agent has identified a number of immunoreactive proteins, including major antigenic proteins of 43 and 45 kDa. Peptides derived from the 43- and 45-kDa proteins were sequenced, and degenerate PCR primers based on these sequences were used to amplify DNA from USG3. Sequencing of a 550-bp PCR product revealed that it encodes a protein homologous to the MSP-2 proteins of Anaplasma marginale. Concurrently, an expression library made from USG3 genomic DNA was screened with granulocytic Ehrlichia (GE)-positive immune sera. Analysis of two clones showed that they contain one partial and three full-length highly related genes, suggesting that they are part of a multigene family. Amino acid alignment showed conserved amino- and carboxy-terminal regions which flank a variable region. The conserved regions of these proteins are also homologous to the MSP-2 proteins of A. marginale; thus, they were designated GE MSP-2A (45 kDa), MSP-2B (34 kDa), and MSP-2C (38 kDa). The PCR fragment obtained as a result of peptide sequencing was completely contained within the msp-2A clone, and all of the sequenced peptides were found in the GE MSP-2 proteins. Recombinant MSP-2B protein and an MSP-2A fusion protein were expressed in Escherichia coli and reacted with human sera positive for the HGE agent by immunofluorescence assay. These data suggest that the 43- and 45-kDa proteins of the HGE agent are encoded by members of the GE MSP-2 multigene family.     

Identification of proteins that are abnormally regulated in differentiated cultured human keratinocytes

The inhabitants living in the neighbourhood of a deserted mercury-contaminated industrial site are subjected to an age-group differentiated mercury exposure assessment based on a scenario-linked calculation. Analytical input data for the calculation procedure are provided for from soil, air and plants in a large number. The most sensitive group are small children being mainly exposed by soil ingestion which makes up nearly 80% of the ADI, followed by inhalation of mercury contaminated indoor air. On the other hand, inhalation of indoor air has a predominant impact on youth and adults.     

Heterogeneous nuclear ribonucleoproteins H, H', and F are members of a ubiquitously expressed subfamily of related but distinct proteins encoded by genes mapping to different chromosomes

Molecular cDNA cloning, two-dimensional gel immunoblotting, and amino acid microsequencing identified three sequence-unique and distinct proteins that constitute a subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins corresponding to hnRNPs H, H', and F. These proteins share epitopes and sequence identity with two other proteins, isoelectric focusing sample spot numbers 2222 (37.6 kDa; pI 6.5) and 2326 (39.5 kDa; pI 6.6), indicating that the subfamily may contain additional members. The identity between hnRNPs H and H' is 96%, between H and F 78%, and between H' and F 75%, respectively. The three proteins contain three repeats, which we denote quasi-RRMs (qRRMs) since they have a remote similarity to the RNA recognition motif (RRM). The three qRRMs of hnRNP H, with a few additional NH2-terminal amino acids, were constructed by polymerase chain reaction amplification and used for ribohomopolymer binding studies. Each qRRM repeat bound poly(rG), while only the NH2-terminal qRRM bound poly(rC) and poly(rU). None of the repeats bound detectable amounts of poly(rA). The expression levels of hnRNPs H and F were differentially regulated in pairs of normal and transformed fibroblasts and keratinocytes. In normal human keratinocytes, the expression level of H was unaffected by treatment with several substances tested including two second messengers and seven cytokines. Likewise the expression level of F was independent of these substances, although it was strikingly down-regulated by long term treatment with 4 beta-phorbol 12-myristate 13-acetate, indicating that the protein kinase C signaling pathway regulates its expression. No effect of 4 beta-phorbol 12-myristate 13-acetate was observed on the expression of hnRNP H. The genes coding for hnRNPs H, H', and F were chromosome-mapped to 5q35.3 (HNRPH1), 6q25.3-q26, and/or Xq22 (HNRPH2) and 10q11.21-q11.22 (HNRPF), respectively.     

Two kinesin light chain genes in mice. Identification and characterization of the encoded proteins

Native kinesin consists of two light chains and two heavy chains in a 1:1 stoichiometric ratio. To date, only one gene for kinesin light chain has been characterized, while a second gene was identified in a genomic sequencing study but not analyzed biochemically. Here we describe new genes encoding kinesin light chains in mouse. One of these light chains is neuronally enriched, while another shows ubiquitous expression. The presence of multiple kinesin light chain genes in mice is especially interesting, since there are two kinesin heavy chain genes in humans (Niclas, J., Navone, F., Hom-Booher, N., and Vale, R. D. (1994) Neuron 12, 1059-1072). To assess the selectivity of kinesin light chain interaction with the heavy chains, we performed immunoprecipitation experiments. The data suggested that the light chains form homodimers with no specificity in their interaction with the two heavy chains. Immunofluorescence and biochemical subfractionation suggested differences in the subcellular localization of the two kinesin light chain gene products. Although both kinesin light chains are distributed throughout the central and peripheral nervous systems, there is enrichment of one in sciatic nerve axons, while the other shows elevated levels in olfactory bulb glomeruli. These results indicate that the mammalian nervous system contains multiple kinesin light chain gene products with potentially distinct functions.     

Molecular analysis of protein domain function encoded by the myb-homologous maize genes C1, Zm 1 and Zm 38

Two maize genes, Zm 1 and Zm 38, related to the regulatory anthocyanin gene C1 were analyzed molecularly and used for fusion constructs in transient domain swapping experiments with the C1 wild-type gene. It was shown that both genes (Zm 1 and Zm 38) influence the expression of the A1 locus, a target gene for C1. Zm 1 activates the A1 promoter, however it does not turn on the whole anthocyanin pathway. The Zm 38 gene product shows functions similar to C1-I, a dominant inhibitor of the C1 wild-type gene. Concerning the trans-inhibition by C1-I two effects seem to be involved, competition for binding and formation of heterodimers. Further analysis of C1 function was carried out by a fine structure analysis of C1 mutants induced by the insertion and excision of transposable elements. These experiments indicate that for the activating domain of the protein, the formation of an alpha helix seems to be more important than a high negative charge.