5-Noncoding sequences have been tabulated for 211 messenger RNAs from higher eukaryotic cells. The 5'-proximal AUG triplet serves as the initiator codon in 95% of the mRNAs examined. The most conspicuous conserved feature is the presence of a purine (most often A) three nucleotides upstream from the AUG initiator codon; only 6 of the mRNAs in the survey have a pyrimidine in that position. There is a predominance of C in positions -1, -2, -4 and -5, just upstream from the initiator codon. The sequence CCAGCCAUG (G) thus emerges as a consensus sequence for eukaryotic initiation sites. The extent to which the ribosome binding site in a given mRNA matches the -1 to -5 consensus sequence varies: more than half of the mRNAs in the tabulation have 3 or 4 nucleotides in common with the CCACC consensus, but only ten mRNAs conform perfectly.

Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs

Conditional mutagenesis in mice has recently been made possible through the combination of gene targeting techniques and site-directed mutagenesis, using the bacteriophage P1-derived Cre/loxP recombination system. The versatility of this approach depends on the availability of mouse mutants in which the recombinase Cre is expressed in the appropriate cell lineages or tissues. Here we report the generation of mice that express Cre in myeloid cells due to targeted insertion of the cre cDNA into their endogenous M lysozyme locus. In double mutant mice harboring both the LysMcre allele and one of two different loxP-flanked target genes tested, a deletion efficiency of 83-98% was determined in mature macrophages and near 100% in granulocytes. Partial deletion (16%) could be detected in CD11c+ splenic dendritic cells which are closely related to the monocyte/macrophage lineage. In contrast, no significant deletion was observed in tail DNA or purified T and B cells. Taken together, LysMcre mice allow for both specific and highly efficient Cre-mediated deletion of loxP-flanked target genes in myeloid cells.

Conditional gene targeting in macrophages and granulocytes using LysMcre mice.

Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research

/pdf/a-promoter-level-mammalian-expression-atlas-2qfe4cltg7.pdf

A promoter-level mammalian expression atlas

Agrobacterium tumefaciens and related Agrobacterium species have been known as plant pathogens since the beginning of the 20th century. However, only in the past two decades has the ability of Agrobacterium to transfer DNA to plant cells been harnessed for the purposes of plant genetic engineering. Since the initial reports in the early 1980s using Agrobacterium to generate transgenic plants, scientists have attempted to improve this “natural genetic engineer” for biotechnology purposes. Some of these modifications have resulted in extending the host range of the bacterium to economically important crop species. However, in most instances, major improvements involved alterations in plant tissue culture transformation and regeneration conditions rather than manipulation of bacterial or host genes. Agrobacterium-mediated plant transformation is a highly complex and evolved process involving genetic determinants of both the bacterium and the host plant cell. In this article, I review some of the basic biology concerned with Agrobacterium-mediated genetic transformation. Knowledge of fundamental biological principles embracing both the host and the pathogen have been and will continue to be key to extending the utility of Agrobacterium for genetic engineering purposes.

/pdf/agrobacterium-mediated-plant-transformation-the-biology-4ez5vf6vl1.pdf

Agrobacterium-Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

Analysis of Development Edited by Prof Benjamin H Willier, Prof Paul A Weiss and Prof Viktor Hamburger Pp xii + 735 (Philadelphia and London: W B Saunders Company, 1955) 105s

Mechanisms of Development

A primer-dependent poly (A)-polymerizing activity using ATP as substrate (ATP: RNA adenyltransferase) was isolated in high yield from Escherichia coli and purified to apparent homogeneity. For this purpose an assay system had to be used which restricted a variety of infering enzyme activities. Since the enzyme aggregates to macromolecular cell components or precipitates when kept in low salt conditions (<0.4 M NaCl), it was necessary to perform the entire purification procedure in high salt conditions. This was accomplished by using a high salt ribosomal supernatant, a polyethylenglycol-dextran-NaCl phase partition step and a very efficient final high salt phosphocellulose chromatography. At 0.5 M NaCl the enzyme has a molecular weight of approximately 58000. Dodecylsulfate gel electrophoresis shows a single polypeptide chain of molecular weight × 50000. The enzyme has a high preference for ATP as substrate. Manganese ions show higher activity as cofactors than magnesium ions. All classes of natural RNAs are used as primers. The free 3′terminal hydroxyl group of the RNA is required. The enzyme is unable to catalyze a pyrophosphorolysis or a phosphorolysis reaction. It is shown that ATP: RNA adenyl-transferase preferentially synthesizes rather long chains of poly(A) attached to the RNA primers. No relation between the enzyme protein and subunits of the DNA-dependent RNA polymerase could be detected.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1973.tb02953.x

Purification and characterization of adenosine triphosphate: ribonucleic acid adenyltransferase from Escherichia coli.

Unlike in other mammalian species, the major whey protein in mouse is not alpha-lactalbumin, but a cysteine rich, acidic protein with a molecular weight of 14.0 kDa. We have deduced the amino acid sequence of this mouse acidic of whey protein from the nucleotide sequence of cloned cDNA. The positions of the half cysteines suggest that mouse whey acidic protein (WAP) is a two domain protein, very similar in structure to the plant lectin wheat germ agglutinin and the hypothalamic carrier protein neurophysin.

/pdf/mouse-whey-acidic-protein-is-a-novel-member-of-the-family-of-506o3k7hhi.pdf

Mouse whey acidic protein is a novel member of the family of 'four-disulfide core' proteins.

TGGCA-binding proteins are nuclear proteins with high affinity for double-stranded DNA homologous to the prototype recognition sequence 5'YTGGCANNNTGCCAR 3' Their ubiquitous tissue distribution in higher vertebrates characterizes them as a class of highly conserved proteins which may exert a basic function To obtain clues to this function, specific binding sites were mapped on three viral genomes Recognition sites were identified in the enhancer region of the BK virus, in the LTR of the mouse mammary tumor virus, and in the origin of replication of adenovirus 12 The TGGCA-binding protein from HeLa cells appears to be identical to nuclear factor I described by others, which stimulates initiation of adenovirus DNA replication in vitro However, data from MMTV, BKV, and from cellular genes suggest that this specific protein-DNA interaction may also be involved in the control of gene activity

/pdf/the-tggca-protein-binds-to-the-mmtv-ltr-the-adenovirus-3l1ec5ob1t.pdf

The TGGCA protein binds to the MMTV-LTR, the adenovirus origin of replication, and the BK virus enhancer.

Low salt extracts of chicken oviduct nuclei contain a DNA binding protein with high affinity for specific DNA sequences in the flanking regions of the chicken lysozyme gene Two of the three binding sites found within a total of 11 kb upstream from the promoter are located only 92 bp apart from each other Upon comparison of the DNA binding sites, the symmetrical consensus sequence 5'- TGGCANNNTGCCA -3' can be deduced as the protein recognition site This sequence is the central part of 23 to 25 base pairs protected by the DNA binding protein from DNAase I digestion A homologous binding activity can be detected in nuclei from several chicken tissues and from mouse liver

The TGGCA-binding protein: a eukaryotic nuclear protein recognizing a symmetrical sequence on double-stranded linear DNA

https://febs.onlinelibrary.wiley.com/doi/epdf/10.1016/0014-5793%2894%2900585-0

Albrecht E. Sippel

Papers

Purification and characterization of adenosine triphosphate: ribonucleic acid adenyltransferase from Escherichia coli.

Mouse whey acidic protein is a novel member of the family of 'four-disulfide core' proteins.

The TGGCA protein binds to the MMTV-LTR, the adenovirus origin of replication, and the BK virus enhancer.

The TGGCA-binding protein: a eukaryotic nuclear protein recognizing a symmetrical sequence on double-stranded linear DNA

Transcription factor nuclear factor I proteins form stable homo‐ and heterodimers