A model for microtubule-associated protein 4 structure. Domains defined by comparisons of human, mouse, and bovine sequences.

cDNAs encoding human and mouse microtubule-associated protein 4 (MAP 4) were isolated. MAP 4 is encoded by a single gene. Multiple MAP 4 mRNAs are transcribed that are differentially expressed among mouse tissues. Open reading frames for the human and mouse MAP 4 clones indicate three distinct regions consisting of related sequences with different motifs. Approximately 30% of the protein is tandem related repeats of approximately 14 amino acids. Another region contains clusters of serine and proline. Four 18-mer repeats characteristic of the microtubule-binding domains of MAP 2 and tau are located at the carboxyl-terminal portion of MAP 4. Amino acid sequence analysis revealed that human and mouse MAP 4 are homologs of the bovine 190-kDa MAP/MAP U (Aizawa, H., Emori, Y., Murofushi, H., Kawasakai, H., Sakai, H., and Suzuki, K. (1990) J. Biol. Chem. 265, 13849-13855). Mouse and human MAP 4 and the bovine 190-kDa MAP are approximately 75% similar, indicating that these proteins are all members of the same class. Domains with extremely high conservation (greater than or equal to 88%) are: 1) the extreme amino terminus; 2) a proline-rich region between the KDM and S,P domains; 3) the microtubule-binding domain; and 4) the extreme carboxyl terminus.

Role of ABA in Maturation of Rapeseed Embryos.

Development of Brassica napus L. cv Tower embryos of different ages cultured in vitro with and without abscisic acid (ABA) was compared with normal development in situ to investigate the role of ABA in embryo maturation. Endogenous ABA levels were measured by radioimmunoassay, and sensitivity to ABA was assayed in terms of its ability to suppress precocious germination and stimulate accumulation of storage protein and storage protein mRNA. During development in situ, the levels of endogenous ABA and 12S storage protein mRNA both reach their peaks just before the embryos begin to desiccate. The ABA levels during this phase of development also correlate with the time required in culture before germination is evident. Following these peaks, increasing concentrations of exogenous ABA are required to both suppress germination and continue storage protein accumulation in vitro. Thus, both endogenous ABA and ABA sensitivity decline during maturation. The concentrations of exogenous ABA required to suppress germination at these later stages result in abnormally high levels of endogenous ABA and appear to be toxic. These results are consistent with the hypothesis that in maturing rapeseeds, low water content rather than ABA prevents germination during the later stages of development.

Partial immunochemical identity between (Na+ + K+)-ATPase and a membrane component extractable with chloroform: methanol.

An IgG fraction prepared from an antiserum against a holoenzyme preparation of (Na+ + K+)-ATPase precipitated a single antigen when samples of holoenzyme were subjected to crossed immunoelectrophoresis but precipitated an additional, immunochemically-related antigen when a plasma membrane-enriched fraction was subjected to crossed immunoelectrophoresis under the same conditions. The immunochemically-related antigen could be extracted from the plasma membrane fraction with CHCl3:CH3OH.

Nucleotide sequence of a cDNA clone of Brassica napus 12S storage protein shows homology with legumin from Pisum sativum.

The most abundant protein in seeds of Brassica napus (L.) is cruciferin, a legumin-like 12S storage protein. By in vitro translation of embryo RNA, and pulse-chase labelling of cultured embryos with (14)C-leucine, we have shown that the 30 kd α polypeptides and 20 kd ß polypeptides of cruciferin are synthesized as a family of 50 kd precursors which are cleaved post-translationally. One member of the cruciferin family was cloned from embryo cDNA and sequenced. The nucleotide sequence of the cruciferin cDNA clone, pC1, contains one long open reading frame, which originates in a hydrophobic signal peptide region. Therefore, the complete sequence of the cruciferin mRNA was obtained by primer extension of the cDNA. The predicted precursor polypeptide is 488 amino acids long, including the 22 amino acids of the putative signal sequence. The amino acid composition of cruciferin protein is very similar to the predicted composition of the precursor. Comparison with an amino acid sequence of legumin from peas, deduced from the nucleotide sequence of a genomic clone, shows that the α polypeptide precedes the ß polypeptide on the precursor. Cruciferin and legumin share 40% homology in the regions which can be aligned. However, cruciferin contains a 38 amino acid region high in glutamine and glycine in the middle of the α subunit, which is absent in legumin. Legumin has a highly charged region, 57 amino acids long, at the carboxyl-end of the α subunit, which is not found in cruciferin. Both of these regions appear to have originated by reiteration of sequences. re]19850513 ac]19850715.

cDNA clones for Brassica napus seed storage proteins: evidence from nucleotide sequence analysis that both subunits of napin are cleaved from a precursor polypeptide.

Napins are a family of small, basic storage proteins synthesized in Brassica napus (rapeseed) embryos during seed maturation. Cultured embryos also synthesize napins but require exogenous abscisic acid (ABA) to maintain high accumulation rates. We synthesized cDNA from total RNA of embryos cultured on a medium containing ABA, and cloned it into the Pst1 site of pBR322. Two clones containing napin cDNA sequences selected by differential colony hybridization using [32P]cDNA probes from embryos grown with or without ABA were analyzed. These clones, pN1 (insert size = 583 bp) and pN2 (insert size = 739 bp), contained cDNA from two different napin mRNAs. The mRNAs to which they hybridized were found to encode a 21,000-dalton polypeptide that was immunoprecipitated by antibodies to mature napin (subunits of 9,000 and 4,000 daltons). The cDNA clones hybridized to an 850-base mRNA. Nucleotide sequencing demonstrated 95% homology between pN1 and pN2 cDNA inserts and predicted a precursor polypeptide of 178 amino acids, consistent with the 21,000 dalton in vitro translation product. Comparison of the deduced amino acid sequence with published amino acid compositions of mature napin subunits suggests that both the large and the small subunits are present in one precursor polypeptide, and that other regions of the precursor are removed during processing.