PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 9050930

Coding versus intron variability: extremely polymorphic HLA-DRB1 exons are flanked by specific composite microsatellites, even in distant populations.

C Epplen·E J Santos·J F Guerreiro·P van Helden·J T Epplen
View original source ↗Export metadata ↓

Abstract

Although microsatellite typing is the dominant method in genome research and indirect gene diagnosis, precise relationships of exonic and adjacent simple repeat polymorphisms are not known. We investigated exon 2 sequences of HLA-DRB1 genes and their neighbouring (GT)n(GA)m repeats including the intervening single copy spacer. DRB1 is the most polymorphic protein-coding locus in man and all vertebrates investigated. The entire DRB1 variability exists in exon 2. DRB1 genes in different haplotype groups (DR1, DR51, DR52, DR8 and DR53) are accompanied by characteristic modifications of the (GT)n(GA)m block (3' to group-specific single copy spacers). Among more than 520 alleles analysed, > 100 different types of microsatellites were observed. The perfect (GT)n and (GA)m blocks vary in length and may be partly 'degenerated', mostly in a subgroup-specific manner. Interestingly, the extent of microsatellite diversity varies in given DRB1 alleles. While the microsatellites of the DR7, DR9 alleles and in the DR1 group are virtually invariant, in DR4 and DR13, in particular, simple repeats appear hypervariable with at least 15 or 17 different length alleles, respectively. Comparing Caucasians, Bushmen and South American Indians, the microsatellite variation in identical DRB1 alleles (e.g. DRB1*0102, 03011, 1302) is smaller than within any of the DR groups in Caucasians. Taken together, extremely polymorphic DRB1 exons evolve in concert with certain variants of an exceptionally well-preserved microsatellite.

Explore related subjects

Cloning, MolecularDinucleotide RepeatsEscherichia coliEvolution, MolecularExonsHLA-DR AntigensHLA-DRB1 ChainsHumansIntronsMolecular Sequence DataMutationPolymerase Chain ReactionPolymorphism, GeneticJournal ArticleResearch Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

C Epplen, E J Santos, J F Guerreiro, P van Helden, J T Epplen. 1997. Coding versus intron variability: extremely polymorphic HLA-DRB1 exons are flanked by specific composite microsatellites, even in distant populations.. https://doi.org/10.1007/s004390050379

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2027

Molecular Cloning and Reverse Genetics.

This chapter describes a detailed molecular biology protocol for introducing specific point mutations into the chikungunya virus (CHIKV) genome using a reverse genetics strategy. The method utilizes an overlapping PCR-based approach to generate a mutated DNA fragment, which is then cloned into a pre-engineered CHIKV infectious clone plasmid. The protocol covers all major steps, from the initial PCRs to create the mutated insert to its digestion and ligation into the vector. It also includes procedures for bacterial transformation, colony screening via PCR and Sanger sequencing to confirm the mutation, and plasmid purification via miniprep. The document is structured with a clear introduction, a list of all required reagents and equipment, and a step-by-step methods section.

Cloning, Molecular↗
PubMed2027

A Restriction-Free Cloning Approach for Molecular Engineering of Plasmids.

Molecular cloning by PCR amplification using a highly processive, high-fidelity DNA polymerase represents a robust and versatile technique for the precise manipulation of nucleic acid sequences. This approach enables the insertion, replacement, or modification of specific DNA fragments within a cloning vector, thereby generating an accurate copy of a gene or viral segment for downstream applications, such as protein expression, site-directed mutagenesis, and structural or functional analyses. The use of processive, high-fidelity polymerases significantly reduces the occurrence of base substitution errors, ensuring sequence integrity throughout the amplification process. Traditionally, restriction enzymes have been employed to facilitate directional cloning; however, alternative methods allow for mutagenesis without the need for unique and specific restriction sites and can be applied to virtually any cloning or seamless DNA assembly strategy. In this chapter, we describe a restriction enzyme-free and ligation-free PCR-based protocol widely applicable to any circular vector. This method enables targeted mutagenesis of the chikungunya virus (CHIKV) genome, offering a fast, efficient, and reliable strategy for generating mutant constructs suitable for virological and molecular studies.

Cloning, Molecular↗
PubMed2026

Molecular Cloning, Recombinant Expression, and In Silico Structural Analysis of Cu/Zn-Superoxide Dismutase from Trachyspermum ammi.

Superoxide dismutase (SOD) is an essential antioxidant metalloenzyme that is critical for the cellular defense against oxidative damage, as it scavenges superoxide radicals and maintains the redox status. Cytosolic Cu/Zn-SOD is particularly important in the regulation of oxidative stress among different isoforms in higher plants. While Cu/Zn-SODs from several plant species have been characterized, molecular information is limited for Trachyspermum ammi, a medicinally important member of a family Apiaceae with antioxidant potential.In the present study, an integrated molecular and in silico approach has been taken to clone and analyze a Cu/Zn type SOD gene from T. ammi to get insight into its structural and evolutionary characteristics. PCR amplification yielded an open reading frame of 456 bp encoding a protein of 152 amino acids. Sequence analysis showed that plant Cu/Zn-SODs, especially those from Daucus carota, were highly similar to one another (about 90-95%).Multiple sequence alignment confirmed the presence of conserved catalytic motifs and metal-binding histidine residues, both of which are crucial for enzymatic function. Physicochemical analysis predicted the protein to be stable, hydrophilic and compatible with cytosolic localization. The analysis of secondary structure indicated a predominance of β-strands, consistent with the conserved β-barrel architecture of plant Cu/Zn-SODs.The three-dimensional structure was built by homology modeling using a closely related plant Cu/Zn-SOD template with high sequence identity. Structural validation demonstrated an acceptable stereochemical quality with 86.3% residues in the favored region of Ramachandran plot, satisfactory ERRAT and Verify3D scores, and a low RMSD value of 0.104 Å on structural superimposition. Phylogenetic analysis placed the enzyme in the Apiaceae lineage, suggesting evolutionary conservation among related plant species. In conclusion, this study presents the first molecular and structural characterization of Cu/Zn-SOD from T. ammi and confirms the existence of a conserved structural framework typical of plant Cu/Zn-SODs. These results provide a basis for further studies concerning recombinant expression, enzymatic validation and potential relevance in antioxidant and plant stress biology.

Cloning, Molecular↗