Balanced-size and long-size cloning of full-length, cap-trapped cDNAs into vectors of the novel lambda-FLC family allows enhanced gene discovery rate and functional analysis.

We have developed a new class of cloning vectors: lambda-full-length cDNA (lambda-FLC) cloning vectors. These vectors can be bulk-excised for preparing full-length cDNA libraries in which a high proportion of the plasmids carry large inserts that can be transferred into other (for example, functional) vectors. Unlike other cloning vectors, lambda-FLC vectors accommodate a broad range of sizes of eukaryotic cDNA inserts because they contain "size balancers." Further, the main protocol we use for direct bulk excision of plasmids is mediated by a Cre-lox system and is apparently free of size bias. The average size of the inserts from excised plasmid cDNA libraries was 2.9 kb for standard and 6.9 kb for size-selected cDNA. The average insert size of the full-length cDNA libraries was correlated to the rate of new gene discovery, suggesting that effectively cloning rarely expressed mRNAs requires vectors that can accommodate large inserts from a variety of sources. Part of the vectors are also suitable for bulk transfer of inserts into various functional vectors.

Cloning full-length, cap-trapper-selected cDNAs by using the single-strand linker ligation method.

We have developed the single-strand linker ligation method (SSLLM), which uses DNA ligase to add a dsDNA linker to single-stranded (ss) full-length cDNA. The linkers have random 6-bp (dN6 or dGN5) 3' overhangs that can ligate to any cDNA sequence, thereby facilitating the production of cDNA libraries with titers exceeding 1 x 10(6) independent clones. We confirmed that the 5' ends of cDNA inserts cloned by using SSLLM are full-length and include the 5' untranslated regions. The great advantage of our method is that the elimination of the GC tail simplifies the sequencing and protein translation of the full-length clones. Further, our method tags ss cDNAs more efficiently than does the traditional RNA ligase reaction.

Induction of metallothionein after lead administration by three injection routes in mice.

The amount of induced hepatic metallothionein (MT) and the alterations of calcium (Ca) and lead (Pb) concentrations in plasma, liver, kidney, and spleen were compared in male mice after iv, ip, and sc injections of lead acetate at a dose of 30 mg Pb/kg body wt. The amount of hepatic MT at 1 day was in the order of ip greater than iv greater than sc injection approximately 0, despite the hepatic Pb concentration in the order of iv greater than ip greater than sc injection. Heat-stable Pb-binding MT was not detected following any injection route. After the iv injection, a transitory hypercalcemia with hyperphosphatemia was observed. As for the tissue Pb concentration after the iv and ip injections, liver and spleen showed a high concentration, while kidney concentration was relatively low. The high tissue Pb was accompanied by an increase of tissue Ca in most cases. Only 10 to 15% of the total Pb accumulated in the liver at 1 day was recovered from the supernatant fraction after ultracentrifugation. The increase of hepatic Ca was ascribed to that in the sediment fraction. After the sc injection, the tissue Pb concentration was very low and no alterations were observed in tissue Ca concentrations.

Acute renal dysfunction by cadmium injected with cysteine in relation to renal critical concentration of cadmium.

In order to study the critical concentration of cadmium (Cd) in acute renal dysfunction following Cd, male mice were injected IV with Cd complexed with cysteine. The critical concentration was 10 micrograms Cd/g wet weight in whole kidney and it was the same as that for Cd-thionein (Cd-Th), which may suggest that the toxicity of Cd-Th is due to Cd ions liberated from Cd-Th in the kidneys. Renal Cd concentration was at first higher than the critical concentration, but decreased to the critical concentration by 24 h after administration. As an index for renal dysfunction, the uptake of p-aminohippurate (PAH) by renal cortical slices in vitro was sensitive, and showed the different time-course from those of urinary protein and glucose levels. The results suggest the usefulness of PAH uptake as an index. Incidental to the renal dysfunction, renal calcium levels exhibited a marked increase.