β+45 G → C: a novel silent β‐thalassaemia mutation, the first in the Kozak sequence

Abstract

SummaryA family from the Southeast of Italy was found to have a novel b-globin mutant, b+45 G fi C, with the features of a silent b-thalassaemia mutation. It was asymptomatic in two heterozygotes, but its interaction with the severe thalassaemia mutation b-IVS-II-654 C fi T worsened the haematological and biosynthetic phenotype in two compound heterozygotes; moreover, another compound heterozygote, who was also heterozygote for the aaa anti3AE7 , suffered from thalassaemia intermedia. The mutation was found associated in cis with the IVS-II-754 T fi C substitution, which did not lead to abnormally spliced mRNA. Furthermore, the amount of b+45 mRNA was the same as the bA mRNA in the reticulocytes of the carriers. In vitro transcription/translation experiments demonstrated that the b+45 G fi C decreased the efficiency of translation of the b-globin chain by about 30%: this slight impairment was consistent with the observed clinical phenotype. The b+45 G fi C is the first mutation found in the Kozak sequence (GACACCATGG) of the b-globin gene and the first one at the position -6 upstream the ATG. The Kozak consensus sequence plays a major role in the initiation of translation process. The present finding supports the hypothesis that the G in position -6 is important in this process.

Novel exon 1 protein‐coding regions N‐terminally extend human KCNE3 and KCNE4

“…Full-length predicted coding regions each contain adequate Kozak consensus sequences (AUG, and A/G in the 23 position); each also contains a G at the 26 position, which is also thought to facilitate translation initiation (37). Moreover, the previously accepted exon 2 start-site for hKCNE4 is GCCTCAATGCTG, which is considered a weaker Kozak sequence than the newly identified exon 1 start-site, because the former contains neither the G at +4 nor the A/G at 23 (Fig.…”

Characterization of β-defensin 42 expressed in principal cells at the initial segment of the rat epididymis

“…The full-length cDNA of Defb42 with a kozak sequence GCCACC [23] was inserted into the multiple cloning sites (BamHI and XholI) of the eukaryotic expression vector pCMV/tag4. Then, the recombinant expression vector pCMV/tag4-Defb42 was transfected into 293T cells in 10-cm dish with FuGENE ® HD (Roche, Basel, Switzerland) without antibiotics as required by the subsequent antibacterial assay.…”

Hb Foggia or 117(GH5)Phe -> Ser : a new 2 globin allele affecting the Hb-AHSP interaction

“…8 mRNA was purified from reticulocytes of carriers. The ␣2 globin gene cDNA was amplified with RT-PCR 9 and the sequencing revealed the presence of mutated cDNA ( Figure 1B).The semi quantitative analysis of normal/mutated cDNA was carried out with the restriction analysis with the enzyme Ple I, for which the mutation creates a new site 5'-GAGTC(N)4^-3'. The DNA and cDNA PCR amplification was carried out at 20 and 24 cycles on two normal and two heterozygous subjects with the primers (TGAC-CCTCTTCTCTGCACAGCTC)-forward (Gene Bank sequence NG_000006 position 34.315/37) and (GTCT-GAGACAGGTAAACACCTCCAT)-reverse (34.618/42) for genomic DNA (328 bp); (GGCAAGAAGGTGGCC-GACGC)-forward (34.070/89) and (GGGAGGCC-CATCGGGCAGGAGGAAC)-reverse (34.482/506) for cDNA (295 bp).…”