Structural analysis of the alpha N-terminal region of erythroid and nonerythroid spectrins by small-angle X-ray scattering

We used SpalphaI-1-156 peptide, a well-characterized model peptide of the alphaN-terminal region of erythrocyte spectrin, and SpalphaII-1-149, an alphaII brain spectrin model peptide similar in sequence to SpalphaI-1-156, to study their association affinities with a betaI-spectrin peptide, SpbetaI-1898-2083, by isothermal titration calorimetry. We also determined their conformational flexibilities in solution by small-angle X-ray scattering (SAXS) methods. These two peptides exhibit sequence homology and could be expected to exhibit similar association affinities with beta-spectrin. However, our studies show that the affinity of SpalphaII-1-149 with SpbetaI-1898-2083 is much higher than that of SpalphaI-1-156. Our SAXS findings also indicate a significantly more extended conformation for SpalphaII-1-149 than for SpalphaI-1-156. The radius of gyration values obtained by two different analyses of SAXS data and by molecular modeling all show a value of about 25 A for SpalphaI-1-156 and of about 30 A for SpalphaII-1-149, despite the fact that SpalphaI-1-156 has seven amino acid residues more than SpalphaII-1-149. For SpalphaI-1-156, the SAXS results are consistent with a flexible junction between helix C' and the triple helical bundle that allows multiple orientations between these two structural elements, in good agreement with our published NMR analysis. The SAXS findings for SpalphaII-1-149 support the hypothesis that this junction region is rigid (and probably helical) for alphaII brain spectrin. The nature of the junction region, from one extreme as a random coil (conformationally mobile) segment in alphaI to another extreme as a rigid segment in alphaII, determines the orientation of helix C' relative to the first structural domain. We suggest that this particular junction region in alpha-spectrin plays a major role in modulating its association affinity with beta-spectrins, and thus regulates spectrin tetramer levels. We also note that these are the first conformational studies of brain spectrin.     

Globin evolution in the genusXenopus: Comparative analysis of cDNAs coding for adult globin polypeptides ofXenopus borealis andXenopus tropicalis

Summary Globin mRNAs ofXenopus borealis andXenopus tropicalis have been cloned and sequenced. The nucleotide and derived amino acid sequences were compared with each other and with already available data fromXenopus laevis. This analysis rendered clear evidence that the common ancestor ofX. laevis andX. borealis, but not ofX. tropicalis, had lost one amino acid of the -globins prior to a genome duplication event that preceded the segregation of the former two species. Replacement-site substitutions were used to calculate a rough time scale of genome duplication and species segregation. The results suggest an ancient separation between theX. laevis and theX. tropicalis groups occurring approximately 110–120 million years ago. Analysis of the amino acid chains demonstrated various alterations. However, some functional domains, like heme-binding sites and₁₂ contact sites, were subject to a high degree of conservation, indicating the existence of functional constraints on them also in the genusXenopus.     

Nucleotide sequence analysis of three cDNAs coding for Poa p IX isoallergens of Kentucky bluegrass pollen.

Grass pollen allergens are one of the major causes of type I allergic reactions (allergic rhinoconjunctivitis, allergic bronchial asthma, and hayfever) in temperate climates afflicting 15-20% of a genetically predisposed population. Workers have found considerable physico- and immunochemical heterogeneity within the grass pollen allergens which has made them difficult to purify for both therapeutic uses and further biochemical study. We recently reported the construction of a cDNA library in lambda gt11 using mRNA extracted from dehydrated Kentucky bluegrass (KBG, Poa pratensis). Here, we present the nucleotide and deduced amino acid sequences for three KBG pollen allergen cDNA clones, KBG 41, 60, and 31, which were isolated from the above library using a pool of six sera from grass pollen allergic patients. These clones exhibit an exceptionally high degree of sequence similarity to one another, only minor similarity to other known allergens, and no homologies to other known proteins or genes. The predicted molecular mass for the cloned proteins range from 28.3 to 37.8 kDa with pI values of 9.6-10.2. All three clones appear to possess leader peptides and lack asparagine sequons required for N-glycosylation. Therefore, the molecular mass of the post-translationally modified proteins were calculated to be 28.4-34.9 kDa, which is consistent with the size of the polypeptides revealed in Western blots of pollen proteins using an antiserum to a recombinant peptide encoded by the partial cDNA clone KBG 8.3. Northern blotting analysis indicates that expression of the genes corresponding to these clones is confined to pollen tissue. The results suggest that the clones code for a group of proteins that represent a new and previously uncharacterized group of grass pollen isoallergens, which have been hereby designated as Poa p IX.     

Nucleotide sequence of the coding region of the mouse N-myc gene.

A genomic clone for the mouse N-myc gene was isolated and the total nucleotide sequence (4807 bp) of the two coding exons and an intron located between them was determined. The amino acid sequence of the N-myc protein was deduced from the DNA sequence. This protein is composed of 462 amino acids, slightly larger than human and mouse c-myc proteins, and is rich in proline like the c-myc protein. Comparison of the amino acid sequences of the mouse N-myc and c-myc proteins showed that conserved sequences are located in eight regions: four regions are in the N-terminal half of the N-myc protein and are separated from each other by regions poorly homologous to those of the c-myc protein, and the four others are located in the C-terminal half, throughout which certain homology exists. A remarkable sequence containing 13 successive acidic amino acids is present in one of the conserved regions located in the middle of the N-myc protein.     

Molecular cloning and sequence analysis of cDNA containing the entire coding region for human fetal liver cytochrome P-450

From a human fetal liver cDNA library, a cDNA clone (lambda HFL33) containing the entire coding region for a form of cytochrome P-450 related to P-450 HFLa was obtained. The clone was 1,971 bp long and had an open reading frame of 1,509 nucleotides coding for a 503 amino acid polypeptide. The nucleotide and the deduced amino acid sequences of lambda HFL33 were very similar to but clearly distinct from those of NF25 and HLp cDNAs, which code for forms of cytochrome P-450 in adult human liver. The deduced N-terminal amino acid sequence of the HFL33 protein was identical to that of P-450 HFLa.     

Cloning and sequencing of cDNAs coding for the human intra-acrosomal antigen SP-10.

cDNAs coding for the intra-acrosomal protein SP-10 were cloned and characterized as a first step in understanding the expression of this antigen during spermatogenesis. Three overlapping SP-10-specific cDNAs were isolated from a human testes cDNA expression library. These cDNAs hybridized to a 1.35-kb mRNA that was present in human testes but was not found in liver or placenta. Complete sequencing of these cDNAs, designated SP-10-5, SP-10-8, and SP-10-10, produced an 1117-bp sequence containing a 265-amino acid-coding region for the SP-10 protein. Hydrophobicity plots generated from the deduced amino acid sequence showed a very hydrophobic amino terminus characteristic of a signal peptide. Sequence data showed that three different amino acid repeats occurred a total of 16 times in the central third of the SP-10 protein. Interestingly, cDNA SP-10-10 has an internal 57-base pair (19 amino acids) in-frame deletion that is not present in SP-10-5, suggesting that alternative splicing generates more than one SP-10 mRNA. The SP-10 protein appears to be a unique acrosomal protein, based on previous immunohistological data and the observation that SP-10 cDNA sequences did not show any significant homology to other sequences found in the Genbank, National Biomedical Research Foundation, or Swiss sequence banks. A recombinant SP-10 fusion protein was produced in an Escherichia coli expression vector and used to generate a polyclonal antiserum. This antiserum stained the acrosomal cap in situ and reacted with a similar set of peptides on Western blots as did a monoclonal antibody to SP-10.     

Analysis of hepatitis C virus intrahost diversity across the coding region by ultradeep pyrosequencing

Hepatitis C virus (HCV) is the leading cause of liver disease worldwide. In this study, we analyzed four treatment-naïve patients infected with subtype 1a and performed Roche/454 pyrosequencing across the coding region. We report the presence of low-level drug resistance mutations that would most likely have been missed using conventional sequencing methods. The approach described here is broadly applicable to studies of viral diversity and could help to improve the efficacy of direct-acting antiviral agents (DAA) in the treatment of HCV-infected patients.     

Cloning and sequence analysis of the human and Chinese hamster inosine-5'-monophosphate dehydrogenase cDNAs

Inosine-5'-monophosphate dehydrogenase, a key enzyme in the regulation of guanine nucleotide biosynthesis, was purified to homogeneity; and a polyclonal antibody directed against the purified protein was used to isolate human and Chinese hamster IMP dehydrogenase cDNA clones. These clones were sequenced and found to contain an open reading frame of a protein containing 514 amino acids. A sequence of 35 amino acids obtained by analysis of the purified protein is identical to a segment of the protein sequence deduced from the IMP dehydrogenase cDNA. The molecular mass of the deduced protein is 56 kDa, which is the observed molecular mass of the purified protein and of the immunoprecipitated in vitro translation product. Comparison of the protein sequences deduced from the human and Chinese hamster cDNA clones indicates only eight amino acid differences, suggesting that IMP dehydrogenase is a highly conserved protein.     

Cloning and sequence analysis of the human parathyroid hormone gene region

Summary A region of 50 kb around the human PTH gene was cloned and mapped by restriction analysis. Sequence analysis was performed and 3270bp determined, completing the sequence of the gene. The nucleotide sequence was analysed with regard to homology between human, bovine and rat PTH genes, and various potential cis-acting regulatory elements were identified. The gene region lacks an obvious CpG island. The PTH gene region in patients suffering from (pseudo)-hypoparathyroidism was investigated by Southern blotting. No detectable alteration in the fragment patterns was observed. Results of segregation analysis in families with affected individuals was inconclusive.     

Amino acid analysis at the picomole level. Application to the C-terminal sequence analysis of polypeptides.

Amino acids labelled with dimethylaminoazobenzenesulphonyl chloride can be separated by reversed-phase high-pressure liquid chromatography and detected in the visible region (436 nm). All 19 naturally occurring amino acids can be separated on a Zorbax ODS column by employing two different gradient systems consisting of an acetonitrile/aqueous buffer mixture. As little as 2--5 pmol of an individual dimethylaminoazobenzenesulphonyl-amino acid can be quantitatively analysed with reliability, and only 10--30 ng of the dimethylaminoazobenzenesulphonylated protein hydrolysate is needed for each complete amino acid analysis. This new technique is as sensitive as any of the current amino acid analysis methods involving ion-exchange separation plus fluorescence detection, and is technically much simpler. By the combination of this sensitive amino acid-analysing technique with carboxypeptidase, we have been able to determine the C-terminal sequence of polypeptides at the picomole level.