Hemoglobin switching in sheep. Cloning and characterization of the beta A and beta-like embryonic globin genes from genomic DNA.

Analysis of restriction site polymorphisms in the beta-globin gene cluster of a Saudi Arabian female with beta zero-thalassemia demonstrated that both of her beta-globin genes were missing a nonpolymorphic AvaII site in exon 2. Examination of the normal nucleotide sequence surrounding this AvaII site revealed that either of two nucleotide substitutions, TGG----TAG or TGG----TGA, could produce a nonsense codon at codon 37 and eliminate the AvaII site. Consequently, two oligonucleotides (19-mers spanning codons 36 through 41 and containing either TAG or TGA at codon 37) were synthesized and hybridized against genomic DNA of the proband and her family. Specific hybridization with one of the oligomers demonstrated that the patient's beta o-thalassemia was the result of homozygosity for the TGG----TGA mutation at codon 37. In certain cases, oligonucleotide hybridization using genomic DNA may obviate the need for gene cloning and sequencing in the characterization of point mutations.

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Use of oligonucleotide hybridization in the characterization of a beta zero-thalassemia gene (beta 37 TGG----TGA) in a Saudi Arabian family [published erratum appears in Blood 1986 Jul;68(1):323]

Blood ◽

10.1182/blood.v67.4.1185.1185 ◽

1986 ◽

Vol 67 (4) ◽

pp. 1185-1188 ◽

Cited By ~ 20

Author(s):

CD Boehm ◽

CE Dowling ◽

PG Waber ◽

PJ Giardina ◽

HH Jr Kazazian

Keyword(s):

Genomic Dna ◽

Globin Gene ◽

Point Mutations ◽

Saudi Arabian ◽

Globin Genes ◽

Beta Globin ◽

Nucleotide Substitutions ◽

Exon 2 ◽

Oligonucleotide Hybridization

Abstract Analysis of restriction site polymorphisms in the beta-globin gene cluster of a Saudi Arabian female with beta zero-thalassemia demonstrated that both of her beta-globin genes were missing a nonpolymorphic AvaII site in exon 2. Examination of the normal nucleotide sequence surrounding this AvaII site revealed that either of two nucleotide substitutions, TGG----TAG or TGG----TGA, could produce a nonsense codon at codon 37 and eliminate the AvaII site. Consequently, two oligonucleotides (19-mers spanning codons 36 through 41 and containing either TAG or TGA at codon 37) were synthesized and hybridized against genomic DNA of the proband and her family. Specific hybridization with one of the oligomers demonstrated that the patient's beta o-thalassemia was the result of homozygosity for the TGG----TGA mutation at codon 37. In certain cases, oligonucleotide hybridization using genomic DNA may obviate the need for gene cloning and sequencing in the characterization of point mutations.

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MOLECULAR CLONING AND CHARACTERIZATION OF ADENYLOSUCCINATE LYASE cDNA AND GENOMIC DNA IN GRASS CARP

Acta Hydrobiologica Sinica ◽

10.3724/sp.j.1035.2009.61105 ◽

2010 ◽

Vol 33 (6) ◽

pp. 1105-1111

Author(s):

Gang QU ◽

Ji-Rui GU ◽

Wen-Bo GU ◽

Wen-Li ZHU ◽

Jiang WU ◽

...

Keyword(s):

Molecular Cloning ◽

Grass Carp ◽

Genomic Dna ◽

Adenylosuccinate Lyase

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Cloning and Characterization of a Chromosomal Class C β-Lactamase and Its Regulatory Gene in Laribacter hongkongensis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.5.1957-1964.2005 ◽

2005 ◽

Vol 49 (5) ◽

pp. 1957-1964 ◽

Cited By ~ 23

Author(s):

Susanna K. P. Lau ◽

Pak-leung Ho ◽

Maria W. S. Li ◽

Hoi-wah Tsoi ◽

Raymond W. H. Yung ◽

...

Keyword(s):

Genomic Dna ◽

Southern Hybridization ◽

Regulatory Gene ◽

Kinetic Properties ◽

Restriction Fragments ◽

Regulatory Systems ◽

Laribacter Hongkongensis ◽

Consensus Motifs ◽

Class C

ABSTRACT Laribacter hongkongensis, a newly discovered bacterium recently shown to be associated with community-acquired gastroenteritis, is generally resistant to most β-lactams except the carbapenems. We describe the cloning and characterization of a novel chromosomal class C β-lactamase and its regulatory gene in L. hongkongensis. Two genes, ampC and ampR, were cloned by inserting restriction fragments of genomic DNA from L. hongkongensis strain HLHK5 into pBK-CMV to give the recombinant plasmid pBK-LHK-5. The ampR and ampC genes and their promoters were divergently oriented, with the ampR gene immediately upstream of the ampC gene and an intercistronic Lys-R motif, typical of inducible ampC-ampR regulatory systems. The deduced amino acid sequence of the cloned AmpC β-lactamase (pI 8.1) contained consensus motifs characteristic of class C β-lactamases but had identities no greater than 46% to known class C β-lactamases. The kinetic properties of this AmpC were also compatible with those of a class C β-lactamase. PCR of 20 clinical isolates of L. hongkongensis, including HLHK5, showed the presence of both ampC and ampR genes in all isolates. Southern hybridization suggested that the ampC gene of HLHK5 was chromosomally encoded. Subcloning experiments showed that the expression of the ampC gene of HLHK5 was regulated by its ampR gene, which acts as a repressor. The β-lactamase characterized from strain HLHK5 was named LHK-5 (gene, bla LHK-5) and represents the first example of AmpC β-lactamase in the β subdivision of proteobacteria.

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