Rapid cDNA sequencing (expressed sequence tags) from a directionally cloned human infant brain cDNA library

Abstract

A human infant brain cDNA library, made specifically for production of expressed sequence tags (ESTs) was evaluated by partial sequencing of over 1,600 clones. Advantages of this library, constructed for EST sequencing, include the use of directional cloning, size selection, very low numbers of mitochondrial and ribosomal transcripts, short polyA tails, few non-recombinants and a broad representation of transcripts. 37% of the clones were identified, based on matches to over 320 different genes in the public databases. Of these, two proteins similar to the Alzheimer's disease amyloid precursor protein were identified.

Sclerostin: A gem from the genome leads to bone-building antibodies

“…Because these were inactivating mutations, it was clear that this protein, aptly given the name sclerostin, functioned either directly or indirectly as an inhibitor of bone formation. During this same general time period, large-scale cDNA/ expressed sequence tag (EST) (10)(11)(12) and genomic DNA sequencing efforts were being made in academia and industry to rapidly identify new human genes. A novel secreted protein of unknown function, which turned out to be sclerostin, was discovered by computational mining of large DNA sequence databases using either homology-based programs (eg, BLAST) (13,14) or a special CxGxC-class cystine-knot search pattern (C Paszty, unpublished) (15) designed to identify new families of cystine-knot proteins.…”

cDNA cloning of a human 100 kDa de‐ubiquitinating enzyme: the 100 kDa human de‐ubiquitinase belongs to the ubiquitin C‐terminal hydrolase family 2 (UCH2)

“…After transfer to PVDF membranes, bands were sliced out and subjected to automated Edman degradation; 18 partial NHz-terminal sequences were clearly identified. The BLAST search unraveled the presence of an expressed sequence tag (EST) derived from a human brain cDNA library [8]; sequences from 9 peptides were found to match regions of the translated protein sequence predicted from a 1.7 kb cDNA clone, HIBAA66 (Fig. 1).…”

“…Clone RST4 was sequenced on both strands and fused to HIBAA66 clone, thus yielding a 3 kb cDNA clone encoding 803 amino acids. The 5' end of the eDNA was obtained through the 5' anchored PCR technique [8][9][10]; the full length cDNA sequence comprises 3158 bp and a putative AUG codon, which suggest that the cDNA clone encodes a protein of 858 amino acids (calculated Mr = 95,743 Da) with a pI of 4.90 (Fig. 1).…”

“…To this end, partial NH2-terminal sequence determination of peptides derived from the enzyme isolated from human erythrocytes, was carried out. The data allowed us to select one clone from randomly sequenced cDNA from a human brain library [8].…”

Industry benefits from the public funding of intellectual curiosity

“…It is not generally known that we had described this development in 1983 in Nature 1 , preceding by many years the adaptation of the technique to the genome project 2,3 .…”