Sheep α-globin gene sequences: Implications for their concerted evolution and for the down-regulation of the 3′ genes

In sheep as in man and most other mammals, there are two -globin genes (^I and ^II), which are expressed at different levels, the upstream gene being the most efficient. In -globin gene triplication and quadruplication, this trend is confirmed, i.e., the -chain output of the downstream genes progressively decreases. In this study, we have determined the complete sequence of the cDNAs and of both the introns in a triple- haplotype in which each gene could be recognized for the presence of distinct alleles. The sequence analysis reveals that the bodies of the three -globin genes are essentially identical (99.9% homology) and moreover indicates that the down-regulation of additional -globin genes in sheep is not the effect of sequence variation from the Cap to the Poly(A) addition sites. This striking similarity among -genes is higher than that seen in other mammals and is probably sustained by particularly efficient mechanisms of gene conversion and cross-over fixation. Correspondence to: Dr. M.S. Ristaldi     

Tissue-specific expression of mouse α-amylase genes: Nucleotide sequence of isoenzyme mRNAs from pancreas and salivary gland

We have determined the nucleotide sequence of two different mouse α-amylase mRNAs, one found in the pancreas and the other in the salivary gland. The 1577 and 1659 nucleotide mRNAs from pancreas and salivary gland, respectively, are the major α-amylase species which accumulate in each tissue. Differences in mRNA length are primarily in the 5′ noncoding regions. Comparable portions of the mRNAs are 89% homologous. The mRNA sequences predict α-amylase precursor proteins of 508 and 511 amino acid residues, accounting for nearly the entire coding capacity of the mRNAs; differences in protein length occur as a result of a nine nucleotide segment present within the translated portion of salivary gland, but not pancreas, mRNA. The lengths and amino acid compositions of the predicted proteins concur with those determined empirically by others. These proteins differ 12% in amino acid sequence, explaining previously observed differences in net charge and antigenic properties. Finally, translation of the salivary gland α-amylase mRNA is not initiated at the AUG codon nearest the 5′ terminus since that codon is almost immediately followed by the termination triplet UAA. This observation may have implications for the mechanism of translation initiation in eucaroytes.     

Synthesis and Properties of Phosphorylated 3′-O-β-D-Ribofuranosyl-2′-deoxythymidine

Abstract

A strategy was developed for the synthesis of 3′-O-β-D-ribofuranosyl 2′-deoxythymidine derivatives using three different protecting groups, which allows the synthesis of a phosphoramidite building block for oligonucleotide synthesis. Likewise the 5′-O- and 5″-O-phosphorylated analogues were synthesized and their conformation was determined using NMR spectroscopy.     

Chemistry and Anti-HIV Activity of 2′-β-Fluoro-2′,3′-dideoxyguanosine

Abstract

The 2′-β-fluoro analogue of 2′,3′-dideoxyguanosine has been prepared by two synthetic routes. This compound and two analogues have anti-HIV activity in at least two of three host cell systems used (ATH8, CEM, PBL). These compounds, as well as their ddGuo parents, have been characterized with regard to their acid-stabilities, octanol-water partition coefficients, and enzyme substrate properties for adenosine deaminase and purine nucleoside phosphorylase. F-ddGuo analogues are less potent but more stable than their non-fluorinated parent compounds.     

Concise and Efficient Synthesis of 3′-O-Tetraphosphates of 2′-Deoxyadenosine and 2′-Deoxycytidine

We describe concise and efficient synthesis of biologically very important 3′-O-tetraphosphates namely 2′-deoxyadenosine-3′-O-tetraphosphate (2′-d-3′-A4P) and 2′-deoxycytidine-3′-O-tetra-phosphate (2′-d-3′-C4P). N⁶-benzoyl-5′-O-levulinoyl-2′-deoxyadenosine was converted into N⁶-benzoyl-5′-O-levulinoyl-2′-deoxyadenosine-3′-O-tetraphosphate in 87% yield using a one-pot synthetic methodology. One-step concurrent deprotection of N⁶-benzoyl and 5′-O-levulinoyl groups using concentrated aqueous ammonia resulted 2′-d-3′-A4P in 74% yield. The same synthetic strategy was successfully employed to convert N⁴-benzoyl-5′-O-levulinoyl-2′-deoxycytidine into 2′-d-3′-C4P in 68% yield.     

Synthesis and Antiviral Evaluation of 2′,3′-Dideoxy-2′-fluoro-3′-C-hydroxymethyl-β-D-arabinofuranosyl Pyrimidine Nucleosides

Abstract

The synthesis and anti-HBV and anti-HIV activity of a number of 2′,3′-dideoxy-2′-fluoro-3′-C-hydroxymethyl-β-D-arabinofuranosyl pyrimidine nucleosides are reported.     

Synthesis and in vitro antiviral activities of 3′-fluoro (or chloro) and 2′,3′-difluoro 2′,3′-dideoxynucleoside analogs against hepatitis B and C viruses

Chronic infections with hepatitis B virus (HBV) and hepatitis C virus (HCV) lead to serious liver diseases worldwide. Co-infection with HBV and HCV is very common and is associated with increased risk of liver pathogenesis, liver cancer, and liver failure. Several 5-substituted 3′-fluoro (or chloro) (1–4, 6, 7, 17–19) and 2′,3′-difluoro 2′,3′-dideoxynucleosides (15 and 16) were synthesized and evaluated for in vitro antiviral activities against duck hepatitis B virus (DHBV), human hepatitis B virus, and hepatitis C virus. Of these compounds 4, 7, 17, and 19 demonstrated moderate anti-HBV activity, and 2, 4, 7, 8, and 19 were weak inhibitors of HCV. Although 5-iodo derivative (7) was most inhibitory against HCV, it exhibited a reduction in cellular RNA levels in Huh-7 cells. The 5-hydroxymethyl-3′-fluoro-2′,3′-dideoxyuridine (4) and 1-(3-chloro-2,3-dideoxy-β-d-erythro-pentofuranosyl)-5-fluorouracil (19) provided the most inhibition of both viruses without cytotoxicity.     

Chemical-Enzymatic Synthesis of 3′-Amino-2′, 3′-dideoxy-β-D-ribofuranosides of Natural Heterocyclic Bases and Their 5′-Monophosphates

Abstract

Treatment of O², 3′-anhydro-5′-O-trityl derivatives of thymidine (1) and 2′-deoxyuridine (2) with lithium azide in dimethylformamide at 150 °C resulted in the formation of the corresponding isomeric 3′-azido-2′, 3′-dideoxy-5′-O-trityl-β-D-ribofuranosyl N¹- (the major products) and N³-nucleosides (3/4 and 5/6). 3′-Amino-2′, 3′-dideoxy-β-D-ribofuranosides of thymidine [Thd(3′NH₂)], uridine [dUrd(3′NH₂)], and cytidine [dCyd(3′NH₂)] were synthesized from the corresponding 3′-azido derivatives. The Thd(3′NH₂) and dUrd(3′NH₂) were used as donors of carbohydrate moiety in the reaction of enzymatic transglycosylation of adenine and guanine to afford dAdo(3′NH₂) and dGuo(3′NH₂). The substrate activity of dN(3′NH₂) vs. nucleoside phosphotransferase of the whole cells of Erwinia herbicola was studied.     

Studies of the Pharmacokinetics and Toxicology of 2′,3′-Dideoxy-β-L-5-fluorocytidine (β-L-FddC) and 2′,3′-Dideoxy-β-L-cytidine (β-L-ddC) In Vivo; and Synthesis and Antiviral Evaluations of 2′,3′-Dideoxy-β-L-5-azacytidine

Abstract

The pharmacokinetics and toxicology of 2′,3′-dideoxy-β-L-5-fluorocytidine (β-L-FddC) and 2′,3′-dideoxy-β-L-cytidine (β-L-ddC) in mice was investigated. In addition, 2′,3′-dideoxy-β-L-5-azacytidine (β-L-5-aza-ddC) and its α-L-anomer (α-L-5-aza-ddC) were synthesized by coupling the silylated 5-azacytosine derivative with 1-O-acetyl-5-O-(tert-butyldimethylsilyl)-2,3-dideoxy-L-ribofuranose, followed by separation of the α-and β-anomers and were evaluated in vitro against HBV and HIV. β-L-5-aza-ddC was found to show significant anti-HBV activity at approximately the same level as 2′,3′-dideoxy-β-D-cytidine (ddC), which is a known anti-HBV agent. β-L-5-aza-ddC was not cytotoxic to L1210, P388, S-180, and CCRF-CEM cells up to a concentration of 100 μ. Conversely, the α-L-anomer was not active against HBV at the same concentration.     

Analysis and validation of genome-specific DNA variations in 5′ flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes

The thirty-three 5′ flanking conserved sequences of the known low-molecular-weight subunit (LMW-GS) genes have been divided into eight clusters, which was in agreement with the classification based on the deduced N-terminal protein sequences. The DNA polymorphism between the eight clusters was obtained by sequence alignment, and a total of 34 polymorphic positions were observed in the approximately 200 bp regions, among which 18 polymorphic positions were candidate SNPs. Seven cluster-specific primer sets were designed for seven out of eight clusters containing cluster-specific bases, with which the genomic DNA of the ditelosomic lines of group 1 chromosomes of a wheat variety ‘Chinese Spring’ was employed to carry out chromosome assignment. The subsequent cloning and DNA sequencing of PCR fragments validated the sequences specificity of the 5′ flanking conserved sequences between LMW-GS gene groups in different genomes. These results suggested that the coding and 5′ flanking regions of LMW-GS genes are likely to have evolved in a concerted fashion. The seven primer sets developed in this study could be used to isolate the complete ORFs of seven groups of LMW-GS genes, respectively, and therefore possess great value for further research in the contributions of a single LMW-GS gene to wheat quality in the complex genetic background and the efficient selections of quality-related components in breeding programs.     

Analysis and validation of genome-specific DNA variations in 5′ flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes

Glutenins and gliadins are the main components of the wheat storage proteins and make up almost of the proteins found in gluten. Glutenins are polymeric pro-teins whose subunits are held together by in-ter-molecular disulphide bonds. They mainly consist o…