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  • 1
    Electronic Resource
    Electronic Resource
    s.l. : American Chemical Society
    Biochemistry 19 (1980), S. 1644-1650 
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    s.l. : American Chemical Society
    Biochemistry 15 (1976), S. 5506-5511 
    ISSN: 1520-4995
    Source: ACS Legacy Archives
    Topics: Biology , Chemistry and Pharmacology
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Journal of molecular evolution 20 (1984), S. 330-340 
    ISSN: 1432-1432
    Keywords: Zein ; Multigene Families
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Zein cDNA clones were used to study the organization of zein genes within the genome of the inbred maize W64A. When individual clones for the two larger molecular-weight classes of zein proteins (Mr=22,000; Mr=19,000) were used as probes for Southern blot hybridizations of genomic DNA, multiple restriction fragments were found to hybridize. Reconstruction analyses using moderately stringent criteria were used to estimate a total of 70–80 zein sequences within the genome of this inbred maize. The hybridization patterns suggest that zein sequences are clustered within the same restriction fragment. When criteria permitting less cross-hybridization of homologous sequences (Tm-10°C) were used, the banding pattern changed, with some of the bands being reduced in intensity or eliminated entirely. Therefore, by control of hybridization criteria, particular zein genes may be more readily distinguished in a Southern blot analysis. The Southern blot hybridization pattern for the Mr=15,000 zein was less complex. Only a single major band was found, with sufficient hybridization intensity for two or three genes. Genomic Southern analyses of other inbred maizes and related grasses showed similarly complex hybridization patterns with cDNA probes for the 19,000- and 22,000-molecular-weight zeins, suggesting that these sequences have been conserved over evolutionary time. The zein multigene family may therefore have arisen by gene duplication before divergence of the maize, teosinte, andTripsacum species from a common ancestor.
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  • 4
    ISSN: 1432-2048
    Keywords: Avena (seed storage proteins) ; Avenin ; Endoplasmic reticulum ; Globulin ; Protein body ; Storage protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The seed storage proteins of oats (Avena sativa L.) are synthesized and assembled into vacuolar protein bodies in developing endosperm tissue. We used double-label immunolocalization to study the distribution of these proteins within protein bodies of the starchy endosperm. When sections of developing oat endosperm sampled 8 d after anthesis were stained with uranyl acetate and lead citrate, the vacuolar protein bodies consisted of light-staining regions which were usually surrounded by a darker-staining matrix. Immunogold staining of this tissue demonstrated a distinct segregation of proteins within protein bodies; globulins were localized in the dark-staining regions and prolamines were localized in the light-staining regions. We observed two additional components of vacuolar protein bodies: a membranous component which was often appressed to the outside of the globulin, and a granular, dark-staining region which resembled tightly clustered ribosomes. Neither antibody immunostained the membranous component, but the granular region was lightly labelled with the anti-globulin antibody. Anti-globulin immunostaining was also observed adjacent to cell walls and appeared to be associated with plasmodesmata. Immunostaining for both antigens was also observed within the rough endoplasmic reticulum. Based on the immunostaining patterns, the prolamine proteins appeared to aggregate within the rough endoplasmic reticulum while most of the globulin appeared to aggregate in the vacuole.
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  • 5
    ISSN: 1573-5028
    Keywords: maize gene expression ; protoplasts ; transient assays ; transcriptional regulation ; zein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Three DNA regions required for high levels of transcription were identified by transient gene expression analysis of the 5′ flanking region of a 19 kDa α-zein gene. For these analyses, the zein promoter region was fused to the β-glucuronidase (GUS) gene and assayed by transient expression in carrot protoplasts. A 107-bp sequence (-114/-8) containing the TATA box resulted in low levels of GUS activity. Addition of the proximal 75 bp (-189/-114) doubled the level of GUS expression, and a further increase in expression was obtained when additional upstream sequences (-483/-226) were placed 5′ of the zein promoters. Zein upstream sequences enhanced transcription independently of the-189/-114 region. Although the-189/-114 region was not essential for transcription, it was important to obtain maximum GUS activity. A 121 bp upstream sequence (-347/-226) that contains the conserved TGTAAAG sequence gave high levels of GUS activity when placed in either orientation 5′ of the zein promoter sequences. However, nucleotides-347 to-309, containing the TGTAAAG sequence, could be deleted from this fragment without a significant change in GUS activity. Zein upstream sequences did not promote transcription of the GUS gene in somatic maize protoplasts. The upstream activating sequence from the cauliflower mosaic virus (CaMV) 35S promoter placed 5′ of deletion mutants of the zein promoter also failed to produce GUS activity above background.
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Plant molecular biology 18 (1992), S. 827-833 
    ISSN: 1573-5028
    Keywords: α-zein ; gene duplication ; maize genes ; maize pseudogene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have determined the nucleotide sequence of a 7343 bp zein genomic clone (gZ22.8H3) from the maize inbred W64A. Computer-aided analysis of the DNA sequence revealed two contiguous 22 kDa α-zein genes. The 5′ gene (gZ22.8) encodes a complete polypeptide and contains putative regulatory sequences in both the 5′ and 3′ flanking regions that are typical of zein genes. In contrast, the 3′ gene (ψgZ22.8) appears to be a pseudogene, because it contains numerous insertions and deletions that would prevent translation of the mRNA. Alignment of the 5′ and 3′ flanking sequences of both genes indicated that they resulted from a 3.3 kb DNA duplication event.
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Plant molecular biology 17 (1991), S. 309-319 
    ISSN: 1573-5028
    Keywords: DNA-binding proteins ; in vitro binding assay ; promoters ; tissue-specific binding ; transcription factor ; zeins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Promoter regions of alpha- and beta-zein genes were analyzed for binding of nuclear proteins from developing endosperm and seedling tissue of maize. Using a band-shift assay, we identified two distinct protein factors, alpha-1 and beta-1, that interacted specifically with alpha- and beta-zein gene promoter regions, respectively. Alpha-1 was present in nuclei from both endosperm and seedling tissue, whereas beta-1 was found only in nuclei from developing endosperm tissue. Mixing of nuclear extracts demonstrated that seedling tissue contained undetectable amounts of beta-1, rather than having an inhibitor for formation of the beta-1/DNA complex. Chemical footprinting analysis localized the beta-1 recognition site to a 22 bp sequence flanked by CCAT and TATA boxes. The apparent molecular mass of beta-1 was determined to be 29 kDa by southwestern blotting. Based onin vitro binding assays, the greatest concentration of the beta-1 in endosperm nuclei is at 16 days after pollination, which coincides with the time of highest transcriptional activity of the beta-zein gene. These results suggest that beta-1 may act as a tissue-specific,trans-acting regulator of the expression of the beta-zein gene in developing maize endosperm.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Plant molecular biology 23 (1993), S. 825-838 
    ISSN: 1573-5028
    Keywords: endosperm ; lysine ; opaque-2 ; non-zein proteins ; Zea mays ; zeins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The reduction of zein synthesis in the maize (Zea mays L.) opaque-2 mutant is associated with an increased percentage of lysine in the endosperm protein. When expressed on an endosperm basis, we found that W64A opaque-2 contains 490 μg of lysine compared with 350 μg in W64A normal. SDS-PAGE analysis of endosperm proteins indicated that several non-zein proteins are more abundant in the mutant than in normal genotype. To determine the subcellular origin of these proteins, we separated an endosperm homogenate from developing kernels by sucrose density gradient centrifugation and used marker enzyme assays and immunoblot analyses to identify cellular components. Amino acid analysis of proteins in the gradient fractions showed that the majority of the lysine occurs in soluble proteins at the top of the gradient. To identify these proteins, we prepared a complex antiserum against the entire soluble protein fraction and used it to immunoscreen an endosperm cDNA expression library. Sequence analysis of clones identified mRNAs involved in carbohydrate metabolism, amino acid biosynthesis, and protein synthesis. RNA dot blot hybridization analysis with these clones revealed significant variation in the levels of transcripts between normal and opaque-2 endosperm, but we identified several mRNAs that are elevated in opaque-2 and that may encode proteins responsible for the enhanced lysine content.
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  • 9
    ISSN: 1573-5028
    Keywords: cDNA clones ; EST sequencing ; maize ; RFLP mapping
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract As one component of a maize genome project, we report the analysis of a number of randomly selected cDNAs, by a combination of measuring mRNA expression, ‘single-pass’ sequencing (SPS), and genome mapping. Etiolated seedling (490) and membrane-free polysomal endosperm cDNA clones (576) were evaluated for their transcription levels by hybridizing with a probe prepared from total mRNA and categorized as corresponding to abundantly or rarely expressed mRNAs and as either constitutive or tissue-specific. A total of 313 clones from the two libraries were submitted to ‘single-pass’ sequencing from the presumed 5′ end of the mRNA and the nucleotide sequence compared with the GenBank database. About 61% of the clones showed no significant similarities within GenBank, 14% of the clones exhibited a high degree of similarity, while the remaining 25% exhibited a lesser degree of similarity. The chromosomal location of more than 300 clones was determined by RFLP mapping using standard populations. The results demonstrate that a combination of analyses provides synergistic information in eventually deducing the actual function of these types of clones.
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    Plant molecular biology 41 (1999), S. 801-814 
    ISSN: 1573-5028
    Keywords: eEF1A ; endosperm ; gene family ; maize ; opaque2
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract eEF1A appears to be a multifunctional protein in eukaryotes, where it serves as a protein synthesis factor as well as a cytoskeletal protein. In maize endosperm, the eEF1A concentration is highly correlated with lysine content, and eEF1A synthesis is increased in opaque2 mutants compared to wild type. To investigate the basis for the increased synthesis of eEF1A in opaque2, we characterized the genes encoding this protein and measured their relative level of expression in endosperm and other tissues. Maize contains 10 to 15 eEF1A genes that are nearly identical in nucleotide and amino acid sequences. However, these genes can be distinguished based on their 3′ non-coding sequences, which are less conserved. By screening endosperm and seedling cDNA libraries, we show that most of the maize eEF1A genes are expressed, and the relative level of their transcripts varies in different tissues. At least five genes are transcribed in the endosperm, and two account for ca. 80% of the RNA transcripts. The expression of several genes is enhanced in opaque2 endosperm, although the significance of this is unclear.
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