Molecular cloning and sequence analysis of full-length cDNA coding for mouse contrapsin

Four overlapping cDNA clones encoding contrapsin were isolated from a mouse liver cDNA library constructed in the expression vector, lambda gt11. M13 vector sequence analysis revealed that contrapsin cDNA contained an open reading frame of 1,254 bases encoding 418 amino acids. The N-terminal amino acid sequence of the isolated contrapsin matched residues 30 to 48 of the sequence deduced on nucleotide analysis. One clone, which had the longest 3' untranslated region, contained two sets of tandem polyadenylation signals, AATACA and AATAAA, which were located 497 bases apart, while the remaining three clones terminated at the first signal. The entire reading frame sequence of contrapsin cDNA showed 64% homology with that of human alpha-1-antichymotrypsin.     

Nucleotide sequence of a cDNA clone encoding mouse transition protein 1

We have determined the nucleotide sequence of cDNA clones encoding mouse transition protein 1 (TP1), a basic nuclear protein involved in nuclear condensation during spermiogenesis. The nucleotide sequence predicts that transition protein 1 in rats and mice differs by only one amino acid. The rate of substitution of nucleotides in the coding region of mouse and rat transition protein 1 mRNA is close to the average of many proteins in rats and mice, and the usage of degenerate codons is typical of the mouse. The identification of this cDNA clone, in conjunction with previous work (Kleene et al. (1983) Dev. Biol. 98, 455–464; Hecht et al. (1986) Exp. Cell Res. 164, 183–190), demonstrates that the mRNA for mouse transition protein 1 accumulates during the haploid phase of spermatogenesis.     

Conserved cysteine to serine mutation in tyrosinase is responsible for the classical albino mutation in laboratory mice

Albinism, due to a lack of melanin pigment, is one of the oldest known mutations in mice. Tyrosinase (monophenol oxygenase, EC 1.14.18.1) is the first enzyme in the pathway for melanin synthesis, and the gene encoding this enzyme has been mapped to the mouse albino (c) locus. We have used mouse tyrosinase cDNA clones and genomic sequencing to study the albino mutation in laboratory mice. Within the tyrosinase gene coding sequences, a G to C transversion at nucleotide 308, causing a cysteine to serine mutation at amino acid 103, is sufficient to abrogate pigment production in transgenic mice. This same base pair change is fully conserved in classical albino strains of laboratory mice. These results indicate that a conserved mutation in the tyrosinase coding sequences is responsible for the classical albino mutation in laboratory mice, and also that most albino laboratory mouse strains have been derived from a common ancestor.     

Isolation of a full-length cDNA encoding mouse aromatase P450

A full-length cDNA clone for aromatase P450 has been isolated from a pregnant mouse ovarian cDNA library. The insert of this clone (2394 bp) contains a 1509-bp open reading frame encoding 503 amino acid residues together with a 46-bp 5'-untranslated stretch and an 839-bp 3'-untranslated region to which a poly(A) tract is attached. Northern blot analysis of ovarian RNA from pregnant mice reveals a major mRNA band of 2.5 kb with a minor band of 2.1 kb. Comparison of mouse aromatase P450 with that of rat, human, and chicken shows 91, 81, and 69% identity in the nucleotide sequence and 92, 79, and 69% identity in the deduced amino acid sequence, respectively. The membrane-spanning domain of mouse aromatase P450 is estimated to be an extremely hydrophobic segment located within the N-terminal region of the molecule. Furthermore, a highly conserved heme-binding domain is noticed.     

The GABAA receptor beta 3 subunit gene: characterization of a human cDNA from chromosome 15q11q13 and mapping to a region of conserved synteny on mouse chromosome 7.

A cDNA encoding the human GABAA receptor beta 3 subunit has been isolated from a brain cDNA library and its nucleotide sequence has been determined. This gene, GABRB3, has recently been mapped to human chromosome 15q11q13, the region deleted in Angelman and Prader-Willi syndromes. The association of distinct phenotypes with maternal versus paternal deletions of this region suggests that one or more genes in this region show parental-origin-dependent expression (genetic imprinting). Comparison of the inferred human beta 3 subunit amino acid sequence with beta 3 subunit sequences from rat, cow, and chicken shows a very high degree of evolutionary conservation. We have used this cDNA to map the mouse beta 3 subunit gene, Gabrb-3, in recombinant inbred strains. The gene is located on mouse chromosome 7, very closely linked to Xmv-33 between Tam-1 and Mtv-1, where two other genes from human 15q11q13 have also been mapped. This provides further evidence for a region of conserved synteny between human chromosome 15q11q13 and mouse chromosome 7. Proximal and distal regions of mouse chromosome 7 show genetic imprinting effects; however, the region of homology with human chromosome 15q11q13 has not yet been associated with these effects.     

Cloning and sequence analysis of cDNA for mouse prolactin

The present study was undertaken to find out whether or not sexual dimorphism in biological activities and amino acid compositions of mouse prolactin might be due to heterogeneity in mRNA for mouse prolactin Cloned cDNAs for mouse prolactin were first isolated from a mouse pituitary cDNA library by hybridization with a rat prolactin cDNA. Then, one clone of about 140 positive clones obtained from 2000 transformants was subjected to nucleotide sequence analysis and verified to contain a nearly full length of cDNA sequence coding for mouse prolactin precursor. The deduced complete amino acid sequence indicates that the precursor molecule consists of 31 amino acids as the signal peptide and 197 amino acids of prolactin, in which two amino acids were found to be different from the amino acid sequence previously published elsewhere. S1 nuclease mapping analysis using male and female pituitary RNAs indicates that mouse preprolactin is encoded by two mRNAs in both sexes. The two mRNAs differ from each other based upon the deletion of three nucleotides in the coding region for the signal peptide determined by the nucleotide sequence analysis in other cDNA clones. In the present study, no sexual difference was revealed in murine prolactin mRNA.     

Molecular cloning and nucleotide sequence of full-length cDNA for sweet potato catalase mRNA

A nearly full-length cDNA clone for catalase (pCAS01) was obtained through immunological screening of cDNA expression library constructed from size-fractionated poly(A)-rich RNA of wounded sweet potato tuberous roots by Escherichia coli expression vector-primed cDNA synthesis. Two additional catalase cDNA clones (pCAS10 and pCAS13), which contained cDNA inserts slightly longer than that of pCAS01 at their 5'-termini, were identified by colony hybridization of another cDNA library. Those three catalase cDNAs contained primary structures not identical, but closely related, to one another based on their restriction enzyme and RNase cleavage mapping analyses, suggesting that microheterogeneity exists in catalase mRNAs. The cDNA insert of pCAS13 carried the entire catalase coding capacity, since the RNA transcribed in vitro from the cDNA under the SP6 phage promoter directed the synthesis of a catalase polypeptide in the wheat germ in vitro translation assay. The nucleotide sequencing of these catalase cDNAs indicated that 1900-base catalase mRNA contained a coding region of 1476 bases. The amino acid sequence of sweet potato catalase deduced from the nucleotide sequence was 35 amino acids shorter than rat liver catalase [Furuta, S., Hayashi, H., Hijikata, M., Miyazawa, S., Osumi, T. & Hashimoto, T. (1986) Proc. Natl Acad. Sci. USA 83, 313-317]. Although these two sequences showed only 38% homology, the sequences around the amino acid residues implicated in catalytic function, heme ligand or heme contact had been well conserved during evolution.     

Human hypoxanthine-guanine phosphoribosyltransferase: a single nucleotide substitution in cDNA clones isolated from a patient with Lesch-Nyhan syndrome (HPRTMidland)

We have determined the molecular basis for hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency in a patient, J.H., with Lesch-Nyhan syndrome. Radioimmunoassay of lysates of erythrocytes or cultured B-lymphoblasts showed that this patient had no detectable HPRT enzyme activity or HPRT protein. HPRT-specific mRNA levels were normal by Northern analysis. We created a cDNA library from mRNA isolated from cultured lymphoblasts derived from this patient. Nucleotide sequencing of full-length HPRT cDNA clones revealed a single nucleotide (nt) substitution: a T-to-A transversion at nt 389. We have designated this variant HPRTMidland. The predicted amino acid (aa) substitution in HPRTMidland is a valine to aspartic acid at aa 130. This substitution is within 2 aa of the amino acid substitution in a previously defined HPRT variant, HPRTAnn Arbor. Both mutations are within a highly conserved sequence in the putative 5-phosphoribosyl-1-pyrophosphate-binding domain. The amino acid substitution in HPRTMidland causes a significant perturbation in the predicted secondary structure of this region. The HPRTMidland mutation affects a different domain of HPRT than the HPRTFlint mutation located at 167 nt away.     

Extensive polymorphism in the extracellular domain of the mouse B cell differentiation antigen Lyb-2/CD72.

Lyb-2/CD72 is a 45-kDa mouse B cell surface protein that binds CD5 and has been shown to play a role in B cell proliferation and differentiation. Using the polymerase chain reaction we have isolated and sequenced cDNA clones encoding the serologically defined mouse Lyb-2a, Lyb-2b, and Lyb-2c alleles. We confirmed that our full length cDNA clones encode the Lyb-2a, -2b, and -2c alleles, respectively, by transfecting the isolated Lyb-2/CD72 cDNA clones into L cells and demonstrating that the transfectants bind only the appropriate allele specific anti-Lyb-2/CD72 antibodies. Sequence comparisons demonstrate that the Lyb-2/CD72 allels are highly conserved in their cytoplasmic and transmembrane domains but exhibit a high degree of polymorphism in their extracellular domains. This polymorphism in the extracellular region involves amino acid substitutions at a minimum of 20 residues and is concentrated primarily in the membrane distal region. cDNA sequence comparisons also demonstrate two distinct seven amino acid insertion/deletions among these allelic variants. A form of Lyb-2b cDNA lacking the sequence encoding the transmembrane region was isolated from a C57B1/6 mouse and a CH12.LX subline. The Lyb-2/CD72 PCR products from mRNA of mice expressing Lyb-2a and Lyb-2c contain a DNA fragment that corresponds in size to the transmembraneless form, suggesting that these mouse strains also express this mRNA.     

cDNA structure of murine C4b-binding protein, a regulatory component of the serum complement system

A cDNA library representing total poly(A+) RNA from the livers of male B10.WR mice was screened with a 1097 base pair (bp) probe obtained from a partial human C4b-binding protein (C4BP) cDNA clone. Two cDNA clones were isolated, the largest of which was sequenced and found to be 1889 bp in length exclusive of the poly(A) tail. The predicted mouse C4BP polypeptide chain encoded by 1239 bp is 413 amino acid residues in length and has a calculated molecular weight of 45,281. The 370-nucleotide sequence upstream from the codon for the predicted amino terminus contains two possible in-phase translational start signals which yield leader sequences of 56 and 13 amino acid residues, respectively. The 3'-untranslated region is 277 bp long, and there are two potential overlapping poly(A) recognition signals, AATTAA and ATTAAAA, located 26 and 25 bp, respectively, upstream from the poly(A) tail; these are preceded by five other potential polyadenylation signals. Beginning at the amino terminus and continuing through to residue 358, there are six contiguous regions of internal homology, each about 60 amino acids in length. The carboxy-terminal 55 amino acid sequence shares no homology with the repeating units. Extensive homology was found with human C4BP at the amino acid level (61%) as well as at the nucleotide level for both the coding and 3'-untranslated regions. Significant differences, however, were observed between mouse and human C4BP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mapping of sequences required for mouse neurovirulence of poliovirus type 2 Lansing.

Intracerebral inoculation of mice with poliovirus type 2 Lansing induces a fatal paralysis, while most other poliovirus strains are unable to cause disease in the mouse. To determine the molecular basis for Lansing virus neurovirulence, we determined the complete nucleotide sequence of the Lansing viral genome from cloned cDNA. The deduced amino acid sequence was compared with that of two mouse-avirulent strains. There are 83 amino acid differences between the Lansing and Sabin type 2 strain and 179 differences between the Lansing and Mahoney type 1 strain scattered throughout the genome. To further localize Lansing sequences important for mouse neurovirulence, four intertypic recombinants were isolated by exchanging DNA restriction fragments between the Lansing 2 and Mahoney 1 infectious poliovirus cDNA clones. Plasmids were transfected into HeLa cells, and infectious recombinant viruses were recovered. All four recombinant viruses, which contained the Lansing capsid region and different amounts of the Mahoney genome, were neurovirulent for 18- to 21-day-old Swiss-Webster mice by the intracerebral route. The genome of neurovirulent recombinant PRV5.1 contained only nucleotides 631 to 3413 from Lansing, encoding primarily the viral capsid proteins. Therefore, the ability of Lansing virus to cause paralysis in mice is due to the viral capsid. The Lansing capsid sequence differs from that of the mouse avirulent Sabin 2 strain at 32 of 879 amino acid positions: 1 in VP4, 5 in VP2, 4 in VP3, and 22 in VP1.     

Analysis of several bovine lutropin beta subunit cDNAs reveals heterogeneity in nucleotide sequence

Several, independent lutropin beta subunit cDNA clones have been isolated from a lambda gt-11 pituitary cDNA library. The complete nucleotide sequence of four clones was determined. A number of single nucleotide differences were detected including three changes within the coding sequence. One of these nucleotide changes resulted in a change in the predicted amino acid sequence, while two did not. These changes may reflect heterogeneity in the animals from which pituitaries were obtained, or they may reflect errors which occurred during the construction or cloning of the cDNA. Two other differences in nucleotide sequence suggest differences in the processing of precursor mRNAs. One difference involves the deletion of three nucleotides from the coding sequence of one of the clones. This probably reflects the use of alternate splicing sites at intron-exon boundaries. The other likely processing difference is reflected by the fact that one of the clones contains a much longer 3' untranslated region than the other clones. This difference probably arose due to a single nucleotide difference leading to selection of an alternate polyadenylation site.     

Isolation of several cDNAs encoding yeast peroxisomal enzymes

Several candidate clones carrying partial cDNAs for yeast peroxisomal enzymes, such as catalase, carnitine acetyltransferase, isocitrate lyase, malate synthase and acyl-CoA oxidase, were efficiently isolated at a single plating from a phage lambda gt11 recombinant cDNA library prepared with poly(A)-rich RNA from an n-alkane-grown yeast, Candida tropicalis, with a mixture of antibodies against the respective purified enzymes. Among them, one candidate clone carrying partial cDNA for catalase was subcloned and subjected to nucleotide sequence analysis. We succeeded in determining that the amino acid sequence deduced from the nucleotide analysis included the sequences derived from the two peptide fragments obtained from the purified enzyme.     

Acatalasemia

Summary The abnormalities in acatalasemia at the gene level as well as properties of the residual catalase in Japanese acatalasemia are historically reviewed. The replacement of the fifth nucleic acid, guanine, in the fourth intron by adenine in the acatalasemic gene causes a splicing mutation and hence a deficiency of mRNA. The guanine-to-adenine substitution was detected in two Japanese acatalasemic cases from different families. The properties of the residual catalase are similar to those of normal catalase; the exons are identical. The properties of the residual catalase and the molecular defect in the catalase gene are compared among Japanese, Swiss, and mouse acatalasemias. The physiological role of catalase, as judged from human acatalasemic blood and acatalasemic mice, is also described.     

Primary structure of a genomic zein sequence of maize.

The nucleotide sequence of a genomic clone (termed Z4 ) of the zein multigene family was compared to the nucleotide sequence of related cDNA clones of zein mRNAs. A tandem duplication of a 96-bp sequence is found in the genomic clone that is not present in the related cDNA clones. When the duplication is disregarded, the nucleotide sequence homology between Z4 and its related cDNAs was approximately 97%. The nucleotide sequence is also compared to other isolated cDNAs. No introns in the coding region of the zein gene are detected. The first nucleotide of a putative TATA box, TATAAATA , was located 88 nucleotides upstream of the first nucleotide of the first ATG codon which initiated the open reading frame. The first nucleotide of a putative CCAAT box, CAAAAT , appeared 45 nucleotides upstream of the first nucleotide of the zein cDNA clones in the 3' non-coding region also appeared in the genomic sequence at the same locations. The amino acid composition of the polypeptide specified by the Z4 nucleotide sequence is similar to the known composition of zein proteins.     

DSCAM, a highly conserved gene in mammals, expressed in differentiating mouse brain

Down Syndrome Cell Adhesion molecule (DSCAM) is a member of the immunoglobulin superfamily, and represents a novel class of neuronal cell adhesion molecules. In order to understand the cellular functions of DSCAM, we isolated full-length mouse and human cDNA clones, and analysed its expression during mouse development and differentiation. Sequence analysis of the human DSCAM cDNA predicted at least 33 exons that are distributed over 840 kb. When compared to human DSCAM, the mouse homologue showed 90 and 98% identity at the nucleotide and amino acid levels, respectively. In mouse, DSCAM is located on 16C, the syntenic region for human chromosome band 21q22 and also the region duplicated in mouse DS models. DSCAM gene is predicted to encode an approximately 220-kDa protein, and its expression shows dynamic changes that correlate with neuronal differentiation during mouse development. Our results suggest that DSCAM may play critical roles in the formation and maintenance of specific neuronal networks in brain.