Polymorphism in the Ly-17 alloantigenic system of the mouse FcgRII gene.
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Animals↗
Biomedical subjects
B J Morley
Publications and source records attributed to B J Morley.
The embryonic and post-natal expression of the nicotinic receptor alpha 3-subunit in rat lower brainstem.
The mRNA expression of the alpha 3 nicotinic acetylcholine receptor subunit in the rat lower brainstem is more widespread in the embryo and at birth than in the mature brain, but the major cell groups expressing alpha 3 are generally the same, with two exceptions. The transcript for the alpha 3-subunit is transiently expressed in the ventral cochlear nucleus (VCN). Expression was very high in the embryonic and early post-natal VCN, but was significantly decreased in the VCN late in the post-natal period (about post-natal day 15). Conversely, alpha 3 mRNA was not expressed in the motor trigeminal nucleus until about PN3.
Animals↗
Expression of nicotinic acetylcholine receptor mRNA in the adult rat peripheral vestibular system.
The mRNA expression of the neuronal nicotinic acetylcholine receptor subunits was determined in adult rat vestibular end-organs and in Scarpa's ganglion (SCG) by in situ hybridization with [35S] riboprobes. Neurons in the SCG expressed the alpha 4-7 and beta 2-3 mRNAs, but not alpha 3 or beta 4 mRNAs. Not all SCG neurons expressed every mRNA found in SCG. The alpha 6 and beta 2-3 riboprobes labeled all neurons, but alpha 4, alpha 5, and alpha 7 mRNAs were selectively expressed in one or more subpopulations of SCG neurons. Vestibular sensory hair cells, in contrast, expressed only alpha 9 mRNA.
Animals↗
Regional distribution of a creatine transporter in rat auditory brainstem: an in-situ hybridization study.
The expression of an mRNA encoding a creatine transporter (CRT1) was investigated in the rat auditory system under ambient sound conditions, using radiolabeled and non-radiolabeled oligonucleotide in-situ hybridization. The results indicated that CRT1 mRNA is widely distributed in auditory nuclei, including the fusiform and deep layers of the dorsal cochlear nucleus, the ventral cochlear nucleus, the superior olivary complex, the nuclei of the lateral lemniscus and the inferior colliculus. The molecular layer of the dorsal cochlear nucleus and the medial geniculate have low levels of label. Creatine provides cells with a reservoir of high-energy phosphate. Neurons do not synthesize creatine but accumulate it by a transport mechanism, which is probably the limiting step in the regulation of intracellular creatine. Therefore, the quantity of transporter expressed may reflect the utilization of creatine and could serve as an in-vitro indicator of endogenous high-energy metabolism in some cells. Although most auditory nuclei express CRT1 mRNA, the quantity of CRT1 mRNA varies among auditory nuclei, indicating that many auditory nuclei have high and fluctuating energy requirements. The level of CRT1 transcript or protein may be regulated by chronic metabolic changes in the auditory system that may occur, for example, with damage to the acoustic organ or the aging process.
Animals↗
IFN-gamma up-regulates expression of the complement components C3 and C4 by stabilization of mRNA.
We investigated the mechanisms underlying the regulation of complement genes C3 and C4 by IFN-gamma. IFN-gamma (500 U/ml, 24 h incubation) increased steady state mRNA levels for both C3 and C4 in three different cell types (Hep G2, U937, and primary fibroblasts). The response to IFN-gamma in Hep G2 cells was time and dose dependent. At all doses of IFN-gamma and at all incubation times, the transcription rate for these two genes, determined by nuclear run-on assays, was reduced (0.3 +/- 0.1; unstimulated rate = 1.00). The t1/2 of mRNA for C3 and C4 in unstimulated cells was 1.8 +/- 0.3 and 2.2 +/- 0.2 h, respectively. After high-dose IFN-gamma stimulation, both C3 and C4 mRNA levels remained at 100% with respect to baseline at 5 h, but after 12 h, levels fell to 13 +/- 2% (C3) and 8 +/- 3% (C4) of baseline values, giving a half-life for these mRNA species of between 5 and 12 h. IFN-gamma stimulation increased C3 and C4 protein synthesis measured at 24 h. We suggest that it is the increase in mRNA stability that is the major effector mechanism by which IFN-gamma regulates C3 and C4 gene expression.
Cell Line↗
The molecular basis of hereditary complement factor I deficiency.
The molecular basis of hereditary complement factor I deficiency is described in two pedigrees. In one pedigree, there were two factor I-deficient siblings, one of whom was asymptomatic and the other suffered from recurrent pyogenic infections. Their factor I mRNA was analyzed by reverse transcription of fibroblast RNA followed by amplification using the polymerase chain reaction. Both siblings were homozygous for the same transversion (adenine to thymine) at nucleotide 1282 in the cDNA. This mutation causes histidine-400 to be replaced by leucine. The altered histidine is a semi-conserved residue within the serine proteinase family, although no function has been ascribed to it. The proband of the second pedigree studied was found to be a compound heterozygote. One allele had the same mutation as the first family, the second allele had a donor splice site mutation that resulted in the deletion of the mRNA encoded in the fifth exon (a low-density lipoprotein receptor domain) from its transcript.
Alleles↗
Homozygous hereditary C1q deficiency and systemic lupus erythematosus. A new family and the molecular basis of C1q deficiency in three families.
OBJECTIVE: To describe a new kindred with Clq deficiency and to identify the molecular lesions responsible for complete functional C1q deficiency in this and 2 other previously described kindreds. METHODS: The A-, B-, and C-chain genes of C1q were amplified by polymerase chain reaction, cloned, and sequenced. The DNA sequence was checked for mutations. RESULT: Patient 1 had a homozygous G-to-A change at codon 6 of the C chain, causing an amino acid change from Gly to Arg. Patient 2 had a homozygous deletion of a C nucleotide at codon 43 of the C-chain, causing a frame shift, leading to a premature stop codon at codon 108. Patient 3 had a homozygous C-to-T mutation at amino acid position 41 of the C chain, resulting in a premature stop codon. CONCLUSION: In the homozygous state, the mutations are sufficient to cause complete deficiency of Clq. The mutation in patient 1 has been previously reported in a patient of different ethnic origin. A survey of a series of 158 DNA samples from patients with systemic lupus erythematosus showed no other examples of this mutant allele.
Base Sequence↗
New microsatellite polymorphisms identified between C57BL/6, C57BL/10, and C57BL/KsJ inbred mouse strains.
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Animals↗
Localization of mtv44 to the centromeric region of mouse chromosome 11.
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Alleles↗
Molecular basis of hereditary C1q deficiency associated with SLE and IgA nephropathy in a Turkish family.
Two siblings (case 1 and case 2) with homozygous C1q deficiency are described. Both presented with a photosensitive rash, and during follow-up case one developed SLE with nephrotic range proteinuria. Case 2 had microscopic hematuria with a past history of macroscopic hematuria. Renal biopsies revealed mesangioproliferative glomerulonephritis in case 1 and IgA nephropathy in case 2, a new finding in association with C1q deficiency. Since the classical pathway of complement plays a role in the development of antibody responses, the family was also evaluated for the immune response to hepatitis B vaccine. Antibody response to hepatitis B vaccine was normal in both affected members and the rest of the family. The A-, B- and C- chain genes of C1q were amplified by PCR and directly sequenced. A homozygous C to T point mutation was identified in genomic DNA isolated from the patients at codon 186 in the A chain that resulted in a premature stop codon. This mutation was present in both parents and both unaffected sibs in the heterozygous state. This mutation was identical to that previously described in a Slovakian family with C1q deficiency. Because of this finding, a series of 92 genomic DNA samples was screened from ethnically distinct patient groups with SLE to test the hypothesis that this mutation of C1q may be a widespread disease susceptibility gene. No further examples of this mutation were found.
Adolescent↗
Autoantibodies to the collagenous region of C1q occur in three strains of lupus-prone mice.
We have developed an ELISA to measure murine autoantibodies to the collagenous region (CLR) of C1q, using the whole human C1q molecule as the solid-phase ligand, in the presence of 1 M NaCl. The assay was validated by testing positive sera from 20 mice using purified mouse C1q, and from 10 mice using purified human C1q-CLR, as the solid-phase ligands. There were highly significant correlations between results obtained with human C1q (whole molecule) and: (i) mouse C1q (rsp = 0.73, P less than 0.001), and (ii) human Clq-CLR alone (rsp = 0.86, P = 0.001). Antibodies to Clq were measured in 53 MRL/lpr, 17 BXSB and 25 NZB/W lupus-prone mice. Median (range) anti-C1q (CLR) antibody levels in MRL/lpr, BXSB, and NZB/W autoimmune mice aged 3 months were 22 (16-66), 21 (17-39) and 19 (15-27) EU, respectively. The median anti-Clq antibody level in MRL/lpr mice aged 5 months was 76 (35-142) EU, significantly higher than that at 3 months (U = 558, P less than 0.0005). Median anti-C1q antibody level in NZB/W mice at 8 months was 37 (13-74) EU and in BXSB mice at 11 months was 62 (31-231) EU, significantly higher than corresponding values at 3 months (U = 326, and U = 4, P less than 0.001, respectively). This is the first demonstration of anti-C1q (CLR) antibodies in NZB/W and BXSB mice. The pathologic significance and the potential utility of these antibodies for monitoring disease in lupus-prone mice are under evaluation.
Aging↗
The mouse C1q genes are clustered on chromosome 4 and show conservation of gene organization.
Mouse complement component C1q is a serum glycoprotein which consists of six A chains, six B chains and six C chains. The three polypeptides are 223, 228, and 217 residues long, respectively, and are encoded by three genes. DNA probes for mouse C1q A, B, and C chains were hybridized to Southern blots of DNA obtained from various inbred mouse strains. On the basis of fragment length polymorphisms, two different alleles of each of the genes could be identified. The distribution of these alleles was determined in the BXD and LXPL recombinant inbred strain series. Comparison with previously reported strain distribution patterns shows that the genes encoding mouse C1q map to the same locus on distal chromosome 4. Overlapping clones spanning the entire gene cluster of C1q were isolated from genomic libraries using specific cDNA probes. The three genes C1qA, C1qB, and C1qC are closely arranged on a 19 kilobase stretch of DNA in the 5' to 3' orientation A-C-B. Each gene consists of two exons separated by one intron. Sequence comparison of C1q from three different species have shown that the B chains have the strongest similarity. Southern blot analysis of chromosomal DNA from 14 vertebrate species demonstrated highest similarity between the C1qB genes, followed by C1qC and finally C1qA.
Amino Acid Sequence↗
DNase I hypersensitivity mapping and promoter polymorphism analysis of human C4.
Human complement component C4 is encoded by two structurally distinct loci in the major histocompatibility complex (MHC) class III region. The two isotypes, C4A and C4B, differ at only four residues in the C4d fragment, but C4 constitutes the most polymorphic of the complement components. It is not known, however, whether the regions involved in the regulation of C4 expression also display polymorphic variation. By using the technique of DNase I hypersensitivity mapping, we established that the only area of transcriptional activity for C4 in the hepatocyte cell line, HepG2, occurs approximately 500 base pairs upstream of the transcriptional start site. This region was found to be remarkably constant in sequence when analyzed in the context of differing MHC haplotypes including HLA B57, C4A6, C4B1, DR7, which has been correlated with reduced expression of the C4A isotype. Similarly, polymerase chain reaction followed by single-strand conformation polymorphism analysis failed to demonstrate any promoter polymorphisms in 103 individuals comprising 52 systemic lupus erythematosus patients and 51 healthy controls.
Base Sequence↗
Analysis of nicotinic acetylcholine receptor subunits in the cochlea of the mouse.
The present study was designed to catalogue and compare nicotinic receptor subunit messages in the mammalian cochlea. Fourteen nicotinic acetylcholine receptor subunit messages were examined by polymerase chain reaction (PCR) analysis and nucleotide sequencing. Total RNA was extracted from the auditory organs of 14- to 18-day-old CBAJ mice, and mRNA was purified using oligo-dT cellulose. After reverse transcription, resulting cDNA was amplified by PCR with the use of primers specific for the nucleotide sequences representing the following nicotinic receptor subunits: muscle types alpha 1, beta 1, gamma, delta and epsilon and neuronal types alpha 2,3,4,5,6,7 and beta 2,3,4. cDNA from cochlear tissue corresponding to the muscle-type receptor subunit beta 1 and to neuronal-type receptor subunits alpha 2,4,5,6 and beta 2,3 was amplified, whereas cDNA for muscle types alpha 1, gamma, delta and epsilon and neuronal types alpha 3,7 and beta 4 was not. All PCR products were homologous in nucleotide sequence to the corresponding reference cDNAs from which the primers were designed. The current results indicate that nicotinic acetylcholine receptor (nAChR) subunits that are similar or identical to the stated muscle and neuronal types are expressed in the murine cochlea. The presence of messages corresponding to the muscle-type beta 1 and neuronal-type nAChR subunits may be correlated with the atypical cholinergic response of cochlear hair cells to agonists and antagonists.
Animals↗
The organization of the human complement factor I gene (IF): a member of the serine protease gene family.
The human complement factor I gene (IF) was cloned from a flow-sorted cosmid library. The gene spans 63 kb and comprises 13 exons. The first exon, which encodes the leader sequence and 5' untranslated region, is separated from the body of the gene by a large intron of 36 kb. Factor I is a mosaic protein, and there is a correlation between the genomic organization and the modular structure of the protein. The second exon encodes a module found only in complement C6 and C7 (FI/C6/C7); the third and fourth exons encode a single CD5 domain; and the fifth and sixth exons each encode a low-density lipoprotein receptor module. Two very small exons, 21 and 36 bp, then separate the first six exons from the last five that encode the serine protease domain of factor I. Within the serine protease gene family factor I has a unique genomic structure, but it bears a much closer resemblance to trypsin than it does to the other complement system serine proteases, factor B, C2, and C1r/C1s.
Base Sequence↗
Identification of a glutamate/aspartate transporter in the rat cochlea.
The neurotransmitter at the synapses between hair cells and spiral ganglion cells in the cochlea is probably L-glutamate or a similar excitatory amino acid. Glutamate uptake by nerve terminals and glial cells is an important component of neurotransmission at glutamatergic synapses of the central nervous system, for providing a reservoir of transmitter or transmitter precursors and the termination of the released glutamate. Hair cell synapses are not surrounded by glial cells, therefore, the uptake mechanism for glutamate in the cochlea may be unique. cDNA was synthesized from total RNA isolated separately from the rat organ of Corti, spiral ganglia, and lateral wall tissues. The expression of a glutamate/aspartate transporter (GLAST) was detected by DNA amplification with the polymerase chain reaction. The other two members of glutamate transporters in this family were not detected by this method. A partial cDNA encoding to GLAST was identified by sequence analysis in a rat cochlear cDNA library. Data concerning the expression and the molecular structure of the glutamate transporter GLAST in the cochlea may provide important information regarding the neurotransmission process at the hair cell-afferent synapses.
Amino Acid Sequence↗
Solid-phase peptide synthesis and biological activity of bovine thymopoietin II (bTP-II).
Bovine thymopoietin (bTP), a 49 amino acid polypeptide, was synthesized using Merrifield's solid-phase peptide synthesis methodology. The polypeptide was purified using anion-exchange chromatography and reversed-phase HPLC and characterized by mass spectrometry and amino acid analysis of the full-length peptide and of products derived from digestion with Staphylococcus aureus V8 protease. The biological activity of the synthesized product was tested in several assay systems. Synthetic bTP was found to induce the expression of Thy 1.2 antigen on T-lymphocytes from athymic mice, in agreement with previous studies on the biological activity of endogenous bTP. Biological activity at skeletal muscle and neuronal nicotinic acetylcholine receptor sites, as reported by others for bTP, could not be confirmed in our studies. The absence of biological activity at nicotinic receptor sites may be related to the results of a recent report demonstrating the presence of a cobratoxin-like molecule in preparations of natural bTP. These data indicate that synthetic peptides have an important role for the evaluation of the specificity of the biological activity of polypeptides.
Amino Acid Sequence↗
Hereditary complement factor I deficiency.
We describe four cases (from three families) of hereditary factor I deficiency, bringing the total number of cases now reported to 23. In one family there are two affected siblings: one has suffered recurrent pyogenic infections; the other is asymptomatic. In the second family, the patient had recurrent pyogenic infections and a self-limiting vasculitic illness; in the third family, the patient suffered recurrent pyogenic and neisserial infections. All four patients had markedly reduced concentrations of C3 in the serum (family 1 propositus: 28%; family 1 asymptomatic sibling: 15%; family 2: 31%; and family 3: 31% normal human serum) which was in the form of C3b. Low IgG2 levels may occur in primary C3 deficiency, and a reduction in IgG2 concentration to 1.14 g/l (normal: 1.30-5.90 g/l) was found in the patient from family 2. Using radioligand binding assays, we demonstrated increased binding of C3b to erythrocytes in a patient with factor I deficiency. This C3b could not be cleaved by autologous serum but could be cleaved by normal serum or purified factor I. We review and compare the published cases of C3, factor H and factor I deficiency.
Adolescent↗