Noor A. Kalsheker

Molecular characterisation of three alpha-1-antitrypsin deficiency variants: proteinase inhibitor (Pi) nullcardiff (Asp256→Val); Pi Mmalton (Phe51→ deletion) and Pi I (Arg39→Cys)

SummaryThree mutations causing alpha-1-antitrypsin defiiency have been identified by gene amplification and direct DNA sequencing. In the Pi (proteinase-inhibitor) nullcardiff gene, the codon for aspartate at position 256 has mutated to encode valine. In Pi Mmalton and Pi I, the respective mutations are the deletion of the codon for a phenylalanine residue at position 51 or 52, and a single base substitution resulting in arginine being replaced by cysteine at position 39. Examination of the protein tertiary structure suggests that all of these mutations are likely to result in folding abnormalities that may explain the deficiency states.

Acute intermittent porphyria caused by a C→T mutation that produces a stop codon in the porphobilinogen deaminase gene

SummaryA mutation of the porphobilinogen (PBG) deaminase gene that produces the cross-reacting immunological material (CRIM)-negative type of acute intermittent porphyria (AIP) has been identified in one of 43 unrelated patients with this form of the disorder. The mutation is a C→T transition that abolishes a PstI recognition site in exon 9 of the gene and converts a codon for glutamine to a stop codon.

Molecular characterisation of two alpha-1-antitrypsin deficiency variants: proteinase inhibitor (Pi) NullNewport (Gly115→Ser) and (Pi) Z Wrexham (Ser−19→Leu)

SummaryTwo single point mutations in the alpha-1-antitrypsin gene, resulting in AAT deficiency, have been characterised in heterozygotes by DNA amplification and direct sequencing. The mutations result in amino acid substitutions, Gly115→Ser and Ser−19→Leu, in the leader sequence, respectively, and have been designated Pi NullNewport and Pi Z Wrexham. In the two families studied the mutations occur on chromosomes which also carry the common mutation causing Z deficiency. Individuals with such a deficiency are, therefore, compound heterozygotes. It is not known if these particular mutations would only cause a mild form of AAT deficiency in the absence of the Z mutation as they do not appear to cause predictable folding abnormalities. They do, however, result in severe deficiency when the Z mutation occurs in the same gene.

Characterisation of the alpha-1-antitrypsin M3 gene, a normal variant

SummaryBy sequence analysis of the complete proteincoding region of the human alpha-1-antitrypsin gene using polymerase chain reaction techniques, we have characterised one of the normal variants, M3. We have identified a single point mutation between M1 Va1213 and M3 at codon position 376 which is a GAA(Glu) to GAC(Asp) transversion.

Removal of twelve C-terminal amino acids from firefly luciferase abolishes activity

cDNA coding for the luciferase in the firefly Photinus pyralis was cloned using pcDV1 primer and Honjo linker containing SP6 RNA polymerase promoter. This enabled conditions to be established to produce mRNA, capped with m7 GpppG, in vitro and then translated to form light emitting protein. Full length recombinant luciferase produced by in vitro translation, was fully active, had the same isoelectric focusing point as the native enzyme and produced a similar, yellow emission. Removal of the coding sequence for the last 12 amino acids at the C terminus, containing the peroxisome signal peptide, by polymerase chain reaction resulted in greater than or equal to 99% loss in activity of the protein formed from mRNA in vitro. This has important implications for using this luciferase as an indicator or reporter gene in eukaryotic cells, and for identifying the active centre of the enzyme.

Heterozygosity and localisation of normal allelic fragments for an alpha1-antitrypsin homologous sequence

SummaryApproximately 10 kb downstream of the alpha1-antitrypsin (AAT) gene is a homologous sequence. Two polymorphisms detected with the restriction enzymes Bg1II and EcoRI have been reported previously. We describe two additional polymorphisms with the restriction endonucleases TaqI and HindIII and, for all four restriction enzymes, we have mapped the fragments corresponding to the normal alleles in a cosmid clone.