Characterization of cytoplasmic dynein light-intermediate chain isoforms in rat testis.
We obtained three kinds of novel cytoplasmic dynein light-intermediate chain (LIC) isoforms from rat testis and brain by reverse transcription polymerase chain reaction (RT-PCR). The primers for RT-PCR were designed according to LIC-2 (LIC 53/55) (15). In one novel isoform, the 42 bp specific sequence named TDL was inserted between 1,106 and 1,107 nucleotides (nts) of LIC-2, whereas the 57 bp sequence corresponding to LIC-2 1,339-1,395 nts (BDL) was absent. The TDL and BDL regions were specifically digested with restriction enzymatic treatment and followed by subcloning of non-digested cDNA band in testis and brain, producing an isoform without TDL and BDL regions. BDL specific RT-PCR of testis cDNA followed by sequencing produced an isoform with two specific regions. By Northern blot hybridization using TDL and BDL specific antisense oligo DNA probe, 4.4, 3.5, and 2.0 kb of signals were detected. With both TDL and BDL probes, the 2.0 kb signal was intensely detected in testis, while the 4.4 kb was defected in brain. This indicates that TDL and BDL are derived from the same size of mRNAs. In situ hybridization method using these probes showed that all seminiferous epithelial cells, especially late pachytene spermatocytes, were positive, indicating that LIC 53/55 isoforms were coexpressed in these cells. These findings indicate that LIC 53/55 isoforms provide a variety of dynein subunits, and thus may regulate the dynein-dependent intracellular transport system.
Department of Veterinary Anatomy, Graduate School of Agriculture and Life Sciences, University of Tokyo, Japan.
S
S
Maeda
S Y
SY
Nam
M
M
Fujisawa
N
N
Nakamuta
K
K
Ogawa
M
M
Kurohmaru
Y
Y
Hayashi
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry / Q000201:enzymology*; D004398:Dyneins / Q000737:chemistry* / Q000235:genetics; D017403:In Situ Hybridization; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013737:Testis / Q000737:chemistry / Q000201:enzymology*
D007527:Isoenzymes; D012333:RNA, Messenger; D004398:Dyneins