scholarly journals Protein C deficiency Hong Kong 1 and 2: hereditary protein C deficiency caused by two mutant alleles, a 5-nucleotide deletion and a missense mutation

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 126-133 ◽  
Author(s):  
Y Sugahara ◽  
O Miura ◽  
P Yuen ◽  
N Aoki
Keyword(s):  
Amino Acid ◽  
Protein C ◽  
Catalytic Domain ◽  
Stop Codon ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Expression Vectors ◽  
Protein C Deficiency ◽  
Mutant Proteins ◽  
Southern Blot Hybridization Analysis

Abstract We characterized a mutant protein C gene from an individual with no detectable protein C antigen in blood plasma. Southern blot hybridization analysis with human protein C cDNA demonstrated neither gross deletion nor rearrangement of the gene. Sequencing all the exons and exon-intron boundaries of the gene except the 3′ noncoding region showed two mutant alleles. The one, derived from the mother, represents a deletion of 5 nucleotides (nt) (CCCGC) in the end of exon VI (mutation I), predicted to result in the generation of a new stop codon due to a reading frameshift and the premature termination of translation. The other, derived from the father, represents a point mutation (G to A) in exon IX (mutation II), resulting in an amino acid substitution, Gly-376(GGC) to Asp(GAC), in the catalytic domain of the protein. Allele-specific oligonucleotide probe hybridization confirmed the presence of the two mutations. Mutation I would result in a truncated polypeptide of 169 amino acid residues that lacks the heavy chain. Mutation II gives rise to an alteration of a highly conserved amino acid, Gly-376. These data indicate that this patient is a compound heterozygote of the two mutant alleles, each one inherited from each parent. Transient expression assays using COS-7 cells transfected with mutated protein C expression vectors suggested that each of the two mutations leads to the protein C deficiency by causing an impairment of secretion of the respective mutant proteins.

scholarly journals Protein C deficiency Hong Kong 1 and 2: hereditary protein C deficiency caused by two mutant alleles, a 5-nucleotide deletion and a missense mutation

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 126-133 ◽  
Author(s):  
Y Sugahara ◽  
O Miura ◽  
P Yuen ◽  
N Aoki
Keyword(s):  
Amino Acid ◽  
Protein C ◽  
Catalytic Domain ◽  
Stop Codon ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Expression Vectors ◽  
Protein C Deficiency ◽  
Mutant Proteins ◽  
Southern Blot Hybridization Analysis

We characterized a mutant protein C gene from an individual with no detectable protein C antigen in blood plasma. Southern blot hybridization analysis with human protein C cDNA demonstrated neither gross deletion nor rearrangement of the gene. Sequencing all the exons and exon-intron boundaries of the gene except the 3′ noncoding region showed two mutant alleles. The one, derived from the mother, represents a deletion of 5 nucleotides (nt) (CCCGC) in the end of exon VI (mutation I), predicted to result in the generation of a new stop codon due to a reading frameshift and the premature termination of translation. The other, derived from the father, represents a point mutation (G to A) in exon IX (mutation II), resulting in an amino acid substitution, Gly-376(GGC) to Asp(GAC), in the catalytic domain of the protein. Allele-specific oligonucleotide probe hybridization confirmed the presence of the two mutations. Mutation I would result in a truncated polypeptide of 169 amino acid residues that lacks the heavy chain. Mutation II gives rise to an alteration of a highly conserved amino acid, Gly-376. These data indicate that this patient is a compound heterozygote of the two mutant alleles, each one inherited from each parent. Transient expression assays using COS-7 cells transfected with mutated protein C expression vectors suggested that each of the two mutations leads to the protein C deficiency by causing an impairment of secretion of the respective mutant proteins.

Compound Heterozygous Protein C Deficiency Caused by Two Mutations, Arg-178 to Gin and Cys-331 to Arg, Leading to Impaired Secretion of Mutant Protein C

1994 ◽  
Vol 72 (06) ◽  
pp. 814-818 ◽  
Author(s):  
Yuichi Sugahara ◽  
Osamu Miura ◽  
Shinsaku Hirosawa ◽  
Nobuo Aoki
Keyword(s):  
Disulfide Bond ◽  
Protein C ◽  
Catalytic Domain ◽  
Point Mutations ◽  
Expression Vectors ◽  
Compound Heterozygous ◽  
Protein C Deficiency ◽  
Peptide Cleavage ◽  
Mutant Proteins ◽  
Heterozygous Protein C Deficiency

SummaryThe protein C gene in a patient apparently homozygous for protein C deficiency was analyzed. Two different point mutations, each located in a different allele, were detected to reveal that the patient is a compound heterozygote. Mutation of Arg-178 (CGG) to Gin (CAG) [mutation I] was detected in exon VII, in the vicinity of activation peptide cleavage site by thrombin. Mutation of Cys-331 (TGC) to Arg (CGC) [mutation II] was found in exon IX, at one of the sites involved in disulfide bond formation in the catalytic domain of the heavy chain. The alteration of Cys-331 to Arg disables the formation of the disulfide bond and would alter the protein conformation. Transient expression assays using COS-7 cells transfected with protein C expression vectors containing each one of these two mutations suggested that each of the two mutations would lead to the protein C deficiency by an impairment of secretion of the respective mutant proteins.

Application of Two Neutral Mspl DNA Polymorphisms in the Analysis of Hereditary Protein C Deficiency

1990 ◽  
Vol 64 (02) ◽  
pp. 239-244 ◽  
Author(s):  
P H Reitsma ◽  
W te Lintel Hekkert ◽  
E Koenhen ◽  
P A van der Velden ◽  
C F Allaart ◽  
...  
Keyword(s):  
Protein C ◽  
Stop Codon ◽  
Normal Population ◽  
Amino Acid Position ◽  
Rare Allele ◽  
Dna Polymorphisms ◽  
Type I ◽  
Protein C Deficiency ◽  
Gene Deletions ◽  
Allelic Frequencies

SummaryScreening of restriction erzyme digested DNA from normal and protein C deficient individuals with a variety of probes derived from the protein C locus has revealed the existence of two neutral MspI polymorphism. One polymorphism (MI), which is located ≈7 kb upstream of the protein C gene, has allelic frequencies of 69 and 31%, and was used to exclude extensive gene deletions as a likely cause of type I protein C deficiency in 50% of cases in a panel of 22 families. Furtherrnore, the same polymorphism has been used in 5 doubly affected individuals establishing compound heterozygosity in 3 of these.The second, intragenic, polymorphism (MII) has allelic frequencies of 99 and 1% in the normal population. The frequency of the rare allele of this RFLP was with 7% much higher in a panel of 22 Dutch families with protein C deficiency. Interestingly, in all three probands that were heterozygous for MII the rare allele of MII coincided with a point mutation that leads to a stop codon in amino acid position 306 of the protein C coding sequence. This mutation may account for 14% of the protein C deficient individuals in The Netherlands.

Ectopic Transcript Analysis Indicates that Allelic Exclusion is an Important Cause of Type I Protein C Deficiency in Patients with Nonsense and Frameshift Mutations in the PROC Gene

1996 ◽  
Vol 75 (06) ◽  
pp. 870-876 ◽  
Author(s):  
José Manuel Soria ◽  
Lutz-Peter Berg ◽  
Jordi Fontcuberta ◽  
Vijay V Kakkar ◽  
Xavier Estivill ◽  
...  
Keyword(s):  
Splice Site ◽  
Protein C ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Allelic Exclusion ◽  
Polymorphism Analysis ◽  
Type I ◽  
Protein C Deficiency ◽  
Nonsense Mutations ◽  
Stop Codons

SummaryNonsense mutations, deletions and splice site mutations are a common cause of type I protein C deficiency. Either directly or indirectly by altering the reading frame, these' lesions generate or may generate premature stop codons and could therefore be expected to result in premature termination of translation. In this study, the possibility that such mutations could instead exert their pathological effects at an earlier stage in the expression pathway, through “allelic exclusion” at the RNA level, was investigated. Protein C (PROC) mRNA was analysed in seven Spanish type I protein C deficient patients heterozygous for two nonsense mutations, a 7bp deletion, a 2bp insertion and three splice site mutations. Ectopic RNA transcripts from patient and control lymphocytes were analysed by RT-PCR and direct sequencing of amplified PROC cDNA fragments. The nonsense mutations and the deletion were absent from the cDNAs indicating that only mRNA derived from the normal allele had been expressed. Similarly for the splice site mutations, only normal PROC cDNAs were obtained. In one case, exclusion of the mutated allele could be confirmed by polymorphism analysis. In contrast to these six mutations, the 2 bp insertion was not associated with loss of mRNA from the mutated allele. In this case, cDNA analysis revealed the absence of 19 bases from the PROC mRNA consistent with the generation and utilization of a cryptic splice site 3’ to the site of mutation, which would result in a frameshift and a premature stop codon. It is concluded that allelic exclusion is a common causative mechanism in those cases of type I protein C deficiency which result from mutations that introduce premature stop codons

Tandem arrangement of tubulin genes in the protozoan parasite Leishmania enriettii

1983 ◽  
Vol 3 (6) ◽  
pp. 1070-1076
Author(s):  
S M Landfear ◽  
D McMahon-Pratt ◽  
D F Wirth
Keyword(s):  
Tandem Repeat ◽  
Blot Hybridization ◽  
Protozoan Parasite ◽  
Southern Blot Hybridization ◽  
Beta Tubulin ◽  
Developmentally Regulated ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Southern Blot Hybridization Analysis ◽  
Leishmania Enriettii

The arrangement of developmentally regulated alpha- and beta-tubulin genes has been studied in the parasitic protozoan Leishmania enriettii by using Southern blot hybridization analysis. The alpha-tubulin genes occur in a tandem repeat whose monomeric unit may be represented by a 2-kilobase PstI fragment. Similarly, the beta-tubulin genes probably occur in a separate tandem repeat consisting of approximately 4-kilobase units unlinked to the alpha-tubulin repeats.

scholarly journals Tandem arrangement of tubulin genes in the protozoan parasite Leishmania enriettii.

1983 ◽  
Vol 3 (6) ◽  
pp. 1070-1076 ◽  
Author(s):  
S M Landfear ◽  
D McMahon-Pratt ◽  
D F Wirth
Keyword(s):  
Tandem Repeat ◽  
Blot Hybridization ◽  
Protozoan Parasite ◽  
Southern Blot Hybridization ◽  
Beta Tubulin ◽  
Developmentally Regulated ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Southern Blot Hybridization Analysis ◽  
Leishmania Enriettii

The arrangement of developmentally regulated alpha- and beta-tubulin genes has been studied in the parasitic protozoan Leishmania enriettii by using Southern blot hybridization analysis. The alpha-tubulin genes occur in a tandem repeat whose monomeric unit may be represented by a 2-kilobase PstI fragment. Similarly, the beta-tubulin genes probably occur in a separate tandem repeat consisting of approximately 4-kilobase units unlinked to the alpha-tubulin repeats.

A Compound Heterozygous Protein C Deficiency with a Single Nucleotide G Deletion Encoding Gly-381 and an Amino Acid Substitution of Lys for Gla-26

1993 ◽  
Vol 70 (04) ◽  
pp. 636-641 ◽  
Author(s):  
Masaru Ido ◽  
Michiaki Ohiwa ◽  
Tatsuya Hayashi ◽  
Junji Nishioka ◽  
Tsuyoshi Hatada ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein C ◽  
Purpura Fulminans ◽  
Male Infant ◽  
Compound Heterozygous ◽  
Dependent Manner ◽  
Protein C Deficiency ◽  
Single Nucleotide ◽  
Nucleotide Mutation ◽  
Gla Domain

SummaryWe report genetic abnormalities of protein C gene in a male infant who developed neonatal purpura fulminans. DNA-sequence analysis of all exons in protein C gene in this family revealed two mutations. The first abnormality, derived from the mother, was a deletion of one of four consecutive G at nucleotide number 10758 in exon IX which would result in a frame shift mutation and completely change amino acid sequence from Gly381 in the carboxyl-terminal region of protein C. The second abnormality, derived from the father, was a single nucleotide mutation from G to A in the codon (GAG to AAG) at nucleotide number 2977 in exon III, which would result in a substitution of Lys for γ-carboxyglutamic acid (Gla)26. This change would be responsible for the reduced immunological protein C levels of the patient and the father, estimated by a monoclonal antibody which recognizes the Gla-domain in a Ca2+-dependent manner (3.8% and 57%, respectively). Partially purified abnormal protein C from the father’s plasma showed a normal amidolytic activity and a change in the electrophoretic mobility. We detected the above mutations in his family members using two methods; one was a creation of new restriction enzyme sites using mutagenic primers and the other was single nucleotide primer extension. Both methods are rapid and useful for the diagnosis of prenatal protein C abnormalities.

Characterization of the pTFI91 -family replicon of Thiobacillus ferrooxidans plasmids

1995 ◽  
Vol 41 (4-5) ◽  
pp. 354-365 ◽  
Author(s):  
Leena Chakravarty ◽  
Thomas J. Zupancic ◽  
Beth Baker ◽  
Joseph D. Kittle ◽  
Ilona J. Fry ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Thiobacillus Ferrooxidans ◽  
Blot Hybridization ◽  
Marker Gene ◽  
Southern Blot Hybridization ◽  
Structural Features ◽  
Broad Host Range ◽  
Origin Of Replication ◽  
E Coli ◽  
Southern Blot Hybridization Analysis

Plasmids found in six strains of Thiobacillus ferrooxidans were mapped and compared in an effort to detect the origin of replication. Four strains yielded an identical 9.8-kb plasmid, pTFI91. Restriction mapping and Southern blot hybridization analysis were used to confirm this finding. Dissimilar plasmids found in two other strains contained a conserved 2.2-kb SacI region common to pTFI91. DNA sequence analysis of this region showed structural features common to bacterial plasmid replicons. A comparison of the pTFI91 origin with those of T. ferrooxidans pTF-FC2 and other broad host range vectors did not show significant homologous DNA sequences. To verify the replication function, a chloramphenicol acetyl transferase marker gene was ligated at the unique sites of pTFI91, and the plasmid was transformed into Escherichia coli DH5α cells but no transformants were identified. To test the replication of pTFI91 independent of DNA polymerase I in E. coli, different restriction fragments of pTFI91 were cloned into pHSG398 (Cmr, ColEI origin) and transformed into the polA1 mutant SF800, but chloramphenicol-resistant transformants were not detected. Electrotransformation of T. ferrooxidans TFI-70 and Pseudomonas putida ATCC 19151 also failed to yield transformants. The results suggested that the pTFI91 plasmid replicon does not function either in E. coli or in P. putida. Since pTFI91 contains the same origin of replication as other plasmids in several other T. ferrooxidans strains, this replicon may be commonly distributed in T. ferrooxidans.Key words: nucleotide sequence, origin of replication, plasmid DNA, replicon, Thiobacillus ferrooxidans.

Leptospira interrogans serovar sejroe infection in a group of laboratory dogs

1995 ◽  
Vol 29 (3) ◽  
pp. 300-306 ◽  
Author(s):  
E. Scanziani ◽  
L. Crippa ◽  
Anna M. Giusti ◽  
M. Luini ◽  
Maria L. Pacciarini ◽  
...  
Keyword(s):  
Interstitial Nephritis ◽  
Urine Analysis ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Leptospira Interrogans ◽  
Normal Ranges ◽  
Southern Blot Hybridization Analysis ◽  
Antibody Titres ◽  
Renal Infection ◽  
Endonuclease Digestion

Interstitial nephritis was seen histologically in 19 (59%) out of 32 pure-breed beagle dogs (16 males and 16 females) subjected to standard safety tests. In these animals no clinical abnormalities were observed and all the tested parameters (haematology, biochemistry and urine analysis) were within the normal ranges. Leptospiral antibody titres ranging from 1:100 to 1:6400, against a serovar ( hardio) belonging to the Sejroe serogroup, were detected by the microscopic agglutination test (MAT) in the serum of the 19 dogs with interstitial nephritis. All animals without renal lesions were seronegative. Leptospiral antigen was detected immunohistochemically in the kidneys of 4 dogs; leptospires were detected in Warthin-Starry stained sections of one dog. Leptospires were isolated from the kidneys of 3 of the 4 dogs examined by bacterial culture. The isolated strains were typed as serovar sejroe by restriction endonuclease digestion and Southern blot hybridization analysis of their DNA. It was concluded that Leptospira interrogans serovar sejroe, was responsible for an asymptomatic chronic renal infection which was widespread in this group of laboratory dogs.

Sign in / Sign up

Export Citation Format

Share Document