Structure and expression of a cloned cDNA for mouse interferon-beta

A unique sequence in the mouse genome which cross-hybridized to a cloned human interferon-beta 1 gene was detected by DNA blot analysis. Taking advantage of this, a cDNA library prepared from partially purified mRNA for mouse interferon-beta was screened using human interferon-beta 1 DNA as a probe. One of the positive clones, pM beta-3, contained a 680-base pair cDNA insert, whose base sequence contained a single large open reading frame for 182 amino acids. The coding sequences of the cDNA showed homologies of 63% at the nucleotide and 48% at the amino acid level with respect to human interferon-beta 1 cDNA (Taniguchi, T., Ohno, S., Fujii-Kuriyama, Y., and Muramatsu, M. (1980) Gene 10, 11-15). The first 21 amino acids, considered to be the signal peptide, were followed by 24 amino acids, whose sequence was identical with the NH2-terminal sequence that had been reported for mouse interferon-beta from Ehrlich ascites tumor cells (Taira, H., Broeze, R. J., Jayaram, B. M., Lengyel, P., Hunkapiller, M. W., and Hood, L. E. (1980) Science (Wash. D.C.) 207, 528-530). The complete primary sequence of mature interferon-beta polypeptide consisting of 161 amino acids (Mr = 19,700) was deduced. There are three N-glycosylation sites, and this offers an explanation for the larger molecular size (Mr = 26,000-40,000) of natural mouse interferon-beta in comparison to the deduced interferon polypeptide. The cDNA, when fused to a SV40 promoter sequence and then introduced into COS-7 cells, directed the synthesis and secretion of a protein product indistinguishable from the authentic mouse interferon-beta.     

Brain-specific expression of transthyretin mRNA as revealed by cDNA cloning from brain

cDNAs for rat transthyretin mRNA were cloned from a brain cDNA library. Sequencing analyses showed the presence of an additional 5' sequence that had not been reported for the liver mRNA corresponding to the flanking promoter region of the gene. This additional sequence was expressed only in the brain, suggesting the presence of a brain-specific promoter.     

Genetics, evolution, and expression of the 68,000-mol-wt neurofilament protein: isolation of a cloned cDNA probe

A 1.2-kilobase (kb) cDNA clone (NF68) encoding the mouse 68,000-mol-wt neurofilament protein is described. The clone was isolated from a mouse brain cDNA library by low-stringency cross-hybridization with a cDNA probe encoding mouse glial fibrillary acidic protein (Lewis et al., 1984, Proc. Natl. Acad. Sci. USA., 81:2743-2746). The identity of NF68 was established by hybrid selection using mouse brain polyA+ mRNA, and cell-free translation of the selected mRNA species. The cell-free translation product co-migrated with authentic 68,000-mol-wt neurofilament protein on an SDS/polyacrylamide gel, and was immunoprecipitable with a monospecific rabbit anti-bovine neurofilament antiserum. In addition, DNA sequence analysis of NF68 showed 90% homology at the amino acid level compared with the sequence of the porcine 68,000-mol-wt neurofilament protein. At high stringency, NF68 detects a single genomic sequence encoding the mouse 68,000-mol-wt neurofilament protein. Two mRNA species of 2.5 kb and 4.0 kb are transcribed from the single gene in mouse brain. The level of expression of these mRNAs remains almost constant in postnatal mouse brains of all ages and, indeed, in the adult. At reduced stringency, NF68 detects a number of mRNAs that are expressed in mouse brain, one of which encodes the 150,000-mol-wt neurofilament protein. The NF68 probe cross-hybridizes at high stringency with genomic sequences in species as diverse as human, chicken, and (weakly) frog, but not with DNA from Drosophila or sea urchin.     

Norrie disease: Linkage analysis using a 4.2-kb RFLP detected by a human ornithine aminotransferase cDNA probe

Previous study has shown that the usual DNA marker for Norrie disease, the L1.28 probe which identifies the DXS7 locus, can recombine with the disease locus. In this study, we used a human ornithine aminotransferase (OAT) cDNA which detects OAT-related DNA sequences mapped to the same region on the X chromosome as that of the L1.28 probe to investigate the family with Norrie disease who exhibited the recombinational event. When genomic DNA from this family was digested with the PvuII restriction endonuclease, we found a restriction fragment length polymorphism (RFLP) of 4.2 kb in size. This fragment was absent in the affected males and cosegregated with the disease locus; we calculated a lod score of 0.602, at theta = 0.00. No deletion could be detected by chromosomal analysis or on Southern blots with other enzymes. These results suggest that one of the OAT-related sequences on the X chromosome may be in close proximity to the Norrie disease locus and represent the first report which indicates that the OAT cDNA may be useful for the identification of carrier status and/or prenatal diagnosis.     

Identification of an H-2Kd gene using a specific cDNA probe.

A cDNA clone known to code for a mouse histocompatibility (class I) antigen was found to contain a sequence specific for a subpopulation of H-2 genes. This unique sequence is located in the 3'' non-coding region close to the stretch of poly(A) nucleotides. A subclone containing this fragment (pH-2d-5) has been used to select hybridizing mRNA. Translation of the mRNA in vitro shows that H-2Kd mRNA is selected. Southern blot analysis of DNA from congenic recombinant mice show that at least one gene containing this sequence is located at the K locus (region) of the major histocompatibility complex. This gene contains a 3.7-kb BglII and a 13-kb EcoRI restriction endonuclease fragment. This gene has been isolated from a genomic DNA library.     

Detection of a unique human V kappa IV germline gene by a cloned cDNA probe.

We have cloned the cDNA encoding the KIV chain of a human antibody with specificity against the major carbohydrate antigen of Streptococcus A. The cDNA has been used as a genetic probe to estimate the number of germline VKIV genes in human DNA. The presence of unique hybridizing bands on digestion of human DNA with several restriction endonucleases and the equivalence of the DNA in a band to a single gene per haploid genome point to the conclusion that there is a unique human VKIV germline gene. The corollary of this conclusion is that the diversity of human VKIV chains must be exclusively due to somatic mutation. This is supported by examination of the sequences of human KIV chain genes and their KIV chain products. Fusion of the unique germline VKIV gene (1) with one of several JK segments, followed by somatic mutations in the V region of the rearranged KIV gene, can account for the known sequences. The restricted germline gene repertoire may account for the small proportion of human KIV chains in the human K chain sequence library (2).     

Characterization of a cloned cDNA encoding rabbit myelin P2 protein

Myelin P2 is a 14,800-Da cytosolic protein found in rabbit sciatic nerves. It belongs to a family of fatty acid binding proteins and shows a 72% amino acid sequence similarity to aP2/422, the adipocyte lipid binding protein, a 58% sequence similarity to rat heart fatty acid binding protein, and a 40% sequence similarity to cellular retinoic acid binding protein. In order to isolate cDNA clones representing P2, a cDNA library was constructed from poly(A+) RNA isolated from sciatic nerves of 10-day-old rabbit pups. By use of a mixed synthetic oligonucleotide probe based on the rabbit P2 amino sequence, 12 cDNA clones were selected from about 25,000 recombinants. Four of these were further characterized. They contained an open reading frame, which when translated, agreed at 128 out of 131 residues with the known rabbit P2 amino acid sequence. These cDNAs recognize a 1.9-kilobase mRNA present in sciatic nerve, spinal cord, and brain, but not present in liver or heart. The levels of P2 mRNA parallel myelin formation in sciatic nerve and spinal cord with maximal amounts being detected at about 15 postnatal days. This initial study will allow characterization of the P2 gene and its regulation, as well as further studies into the role of P2, the first metabolically active myelin-specific protein to be characterized at the genetic level.