Structure of the mouse serum amyloid P component gene

A genomic DNA clone corresponding to the mouse serum amyloid P component (SAP) has been isolated and characterized for the first time. The numbers of exons, the relative sites of intron/exon junctions, and the size of the coding region for mature SAP protein are all in complete agreement with those of the human SAP gene. In the 5'-flanking region of the mouse SAP gene, there is a small DNA segment (43-base pairs) which is highly homologous with the corresponding region of the human SAP gene. However, most parts of the 5'-flanking regions are not conserved between the mouse and human SAP genes, and several phorbol ester-responsive element-like sequences are present only in the mouse SAP gene.     

Circular-dichroism studies on two murine serum amyloid A proteins.

C.d. studies have shown that mouse SAA2 (serum amyloid A2) protein has about one-half of the alpha-helix content of the SAA1 (serum amyloid A1) analogue (15 as against 32%), although secondary-structure prediction analyses based on sequence data do not suggest such a large difference between the forms. The decreased helical content may be a reflection or indication of a stronger propensity to aggregation of the SAA2 form compared with SAA1. The main elements of secondary structure in both proteins are beta-sheets/turns. Interactions with Ca2+ are accompanied by small losses in alpha-helix content, whereas binding to chondroitin-6-sulphate in the presence of millimolar Ca2+ also decreases the amount of secondary structure. However, SAA2 binding to heparan sulphate increases its beta-sheet structure, whereas with SAA1 secondary structure is not apparently altered by its interaction with heparan sulphate. Computer-generated surface profiles show slight differences in accessibility, hydrophilicity and flexibility between the proteins. Understanding these differences may help to explain why SAA2 is found in amyloid fibrils whereas SAA1 is not. In particular, a stronger tendency to aggregation might be the reason why SAA2 is deposited exclusively in these fibrils.     

Structural diversity of murine serum amyloid A genes. Evolutionary implications

Mouse serum amyloid A (SAA) gene family comprises four members that are closely linked in the chromosome 7. Two of these genes encoding major mouse SAA isotypes (SAA1 and SAA2) are highly homologous not only in exons but also in introns and flanking regions; this sequence homology extends 280 base pairs upstream of major cap sites and 430 base pairs downstream of polyadenylation sites, and the 5' boundary of this homology unit is marked by the CA/GT repeat. Sequence comparison also shows that one (SAA4) of the other two genes is related to the SAA1/2 gene, whereas the other gene (SAA3) evolved independently. Based on these results and the SAA gene arrangement, we discussed mouse SAA gene evolution.     

A new murine Ig VH gene family

A novel murine VH gene family, termed VH10, has been found and characterized. Based on RFLP analysis, this family exhibits extensive polymorphism among inbred strains of mice and encompasses two to five members, depending on the Igh haplotype. Analyses of recombinant inbred strains suggest a map position of this family 5' to the 7183 and Q52 VH gene families. A VH10 gene has been found to encode anti-DNA autoantibodies from lupus mice; another one may be a pseudogene.     

Chromosomal location of the gene encoding the murine acute-phase protein serum amyloid P-component (SAP)

A full-length cDNA clone, pmSAP3, encoding the serum P component (SAP), has been used to search for DNA fragment length variation among mouse strains previously analyzed for differences in endogenous SAP levels. Three alleles were found usingEcoRI-digested DNA. The finding of a single 5.4-kb fragment, alleled, in DNA from DBA/2J mice suggests the presence of a singleSap locus. Segregation of DNA fragment associated withSap ^b andSap ^d alleles was analyzed in three sets of recombinant inbred (RI) strains. The strain distribution pattern found for theSap alleles was identical to that of alleles ofLy-9 in 43 individual RI strains, suggesting tight linkage withLy-9 on mouse chromosome 1. In the BXD RI strains, the SDP of theSap locus, defined by the difference in the endogenous SAP level, is also identical to the SDP of the DNA fragments. We propose to redesignate theSap locus to include both the structural element defined by the DNA polymorphism and the regulatory element involved in the regulation of SAP synthesis. TheSap locus is the major genetic element contributing to the regulation of SAP production. Other genetic factors are also involved, as shown by the presence of nonparental phenotypes in the individual BXH RI strains. This study was performed through special Coordination Funds of the Science and Technology Agency of the Japanese Government and PHS Grant GM24464 to R.W.E.     

The sequence and structure of a new serum amyloid A gene.

The acute phase response is characterized by changes in the serum concentrations of many proteins. A 1000-fold increase in the concentration of serum amyloid A (SAA) protein occurs within 24 hours of LPS injection in the mouse. We have isolated a cDNA clone and its corresponding genomic phage for a third, previously unreported SAA protein. The sequence of the cDNA, the gene's exons and neighboring DNA are presented along with the mapping evidence supporting the gene structure.     

Evolution of the rat kallikrein gene family: Gene conversion leads to functional diversity

Summary Kallikrein-like simple serine proteases are encoded by closely related members of a gene family in several mammalian species. Molecular cloning and genomic Southern blot analysis after conventional and pulsed-field gel electrophoresis indicate that the rat kallikrein gene family comprises 15–20 members, probably closely linked at a single locus. Determination of the nucleotide sequences of the rGK-3,-4, and-6 genes here completes sequence data for a total of nine rat kallikrein family members. Comparison of the rat gene sequences to each other and to those of human and mouse kallikrein family genes reveals patterns of relatedness indicative of concerted evolution. Analysis of nucleotide sequence variants in kallikrein family members shows that most sequence variants are shared by multiple family members; the patterns of shared variants are complex and indicate multiple short gene conversions between family members. Sequence exchanges between family members generate novel assortments of variants in amino acid coding regions that may affect substrate specificity and thereby contribute to the diversity of enzyme activity. Furthermore, small sequence exchanges also may play a role in generating the diverse patterns of tissue-specific expression of rat family members. These analyses indicate an important role for gene conversion in the evolution of the functional diversity of these duplicated genes.     

Gene expression of rat glutathione S-transferases. Evidence for gene conversion in the evolution of the Yb multigene family

We have characterized a cDNA with complete coding sequence for the rat liver glutathione S-transferase subunit 4 (Yb2) isolated from a constructed lambda gt10 cDNA library. Functional expression of the cDNA sequence has resulted in the purification to homogeneity of an enzymatically active anionic glutathione S-transferase. In addition to three previously described Yb-type subunits (Yb1, Yb2, Yb3), we now report characterization of a fourth Yb subunit sequence in the form of a genomic DNA clone lambda GTR15-2. The Yb4 gene has no apparent defect, and the deduced Yb4 polypeptide sequence differs from the other three Ybs by 40 to 53 amino acids. The Yb4 gene organization is similar to that of the Yb2 gene in having a minimum of eight exons. Three out of the seven introns between the two genes are conserved to the extent of more than 88% nucleotide identity. We propose that gene conversion may have played a role in the evolution of these Yb genes.     

Differential induction of the serum amyloid A gene family in response to an inflammatory agent and to amyloid-enhancing factor

The murine serum amyloid A1 (SAA1), SAA2, and SAA3 genes are expressed in various tissues in response to acute inflammation. Prolonged expression may be accompanied by amyloid deposition in liver, spleen, and kidney. Shortly before and during deposition, an amyloid-enhancing factor (AEF) can be extracted from these tissues which accelerates amyloid formation when administered with an inflammatory agent. We have investigated the ability of liver AEF to alter expression of the three SAA genes in liver, spleen, and kidney when administered to normal mice or to mice in which inflammation was created with the injection of silver nitrate. In liver, both AEF and silver nitrate induce SAA1 and SAA2 mRNA accumulation. However, AEF elicits a more rapid response and also acts as a potent inducer of hepatic SAA3 mRNA. Silver nitrate does not induce any SAA mRNA species in kidney, whereas AEF induces all three species. In contrast, AEF induces only SAA3 mRNA in the spleen. We also show that the elevation in hepatic SAA mRNA levels induced by either AEF or silver nitrate is associated with a transient increase in the length of the poly(A) tail.     

Structure of the murine gene encoding apolipoprotein A-I.

A 1.6-kb DNA fragment containing the gene encoding apolipoprotein A-I from the mouse, Mus musculus, has been cloned and sequenced. It contains three exons separated by two introns and encodes a secreted polypeptide of 262 amino acids (aa), 238 of which constitute the mature protein. Comparisons with the rat and human proteins indicate moderate levels of shared identity (71 and 66%, respectively), although the overall aa compositions yield proteins with identical pIs (5.4). Kyte-Doolittle analyses of the three proteins indicate that there is no significant difference in the structure of these apolipoproteins.     

Structure of a human serum amyloid A gene and modulation of its expression in transfected L cells

The structure of a human serum amyloid A (SAA) genomic clone (SAAg9) has been analyzed and the nucleotide sequence of the coding regions is compared with that of the cDNA for apoSAA1. The leader and coding sequences of exons 2 and 3 are identical to SAA1. However, there are 10 nucleotide and 7 derived amino acid substitutions in exon 4. These changes are identical to the amino acid sequence of the amyloid protein associated with familial Mediterranean fever. In particular, the amino acid substitution (Thr to Phe) at residue 69 of SAA1 may have an important role in this type of hereditary amyloidosis. The genomic clone SAAg9 has been transfected into mouse L cells, and constitutive expression of human specific mRNA and protein were observed in stable transfected clones. The expression of both SAA mRNA and protein were increased by incubation of the transfected cells with purified human interleukin-1 (IL-1), both human and mouse recombinant IL-1, and recombinant human tumor necrosis factor alpha. The induction of SAA is pretranslational and is likely to be mediated by protein factor(s) since incubation with cycloheximide diminished IL-1-dependent increase in SAA mRNA.     

Gene silencing analyses against amyloid precursor protein (APP) gene family by RNA interference

Amyloid precursor protein (APP) and amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2) are members of a large gene family. Although APP is known to be the source of the beta-amyloid peptides involved in the development of Alzheimer's disease, the normal functions of APP, APLP1 and APLP2 in cells are poorly understood. In this study, we carried out gene silencing analysis by means of RNA interference with synthetic small interfering RNA duplexes targeting the App, Aplp1 and Aplp2 genes in Neuro2a (N2a) cells, a mouse neuroblastoma cell line. The results demonstrated that cell viability and neurite outgrowth of N2a cells undergoing knockdown of Aplp1 were significantly reduced, compared with N2a cells undergoing knockdown of either App or Aplp2.