I read this book the same weekend that the Packers took on the Rams, and the experience of the latter event, obviously, colored my judgment. Although I abhor anything that smacks of being a handbook (like, \"How to Earn a Merit Badge in Neurosurgery\") because too many volumes in biomedical science already evince a boyscout-like approach, I must confess that parts of this volume are fast, scholarly, and significant, with certain reservations. I like parts of this well-illustrated book because Dr. Sj6strand, without so stating, develops certain subjects on technique in relation to the acquisition of judgment and sophistication. And this is important! So, given that the author (like all of us) is somewhat deficient in some areas, and biased in others, the book is still valuable if the uninitiated reader swallows it in a general fashion, realizing full well that what will be required from the reader is a modulation to fit his vision, propreception, adaptation and response, and the kind of problem he is undertaking. A major deficiency of this book is revealed by comparison of its use of physics and of chemistry to provide understanding and background for the application of high resolution electron microscopy to problems in biology. Since the volume is keyed to high resolution electron microscopy, which is a sophisticated form of structural analysis, but really morphology in a modern guise, the physical and mechanical background of The instrument and its ancillary tools are simply and well presented. The potential use of chemical or cytochemical information as it relates to biological fine structure , however, is quite deficient. I wonder when even sophisticated morphol-ogists will consider fixation a reaction and not a technique; only then will the fundamentals become self-evident and predictable and this sine qua flon will become less mystical. Staining reactions (the most inadequate chapter) ought to be something more than a technique to selectively enhance contrast of morphological elements; it ought to give the structural addresses of some of the chemical residents of cell components. Is it pertinent that auto-radiography gets singled out for more complete coverage than other significant aspects of cytochemistry by a high resolution microscopist, when it has a built-in minimal error of 1,000 A in standard practice? I don't mean to blind-side (in strict football terminology) Dr. Sj6strand's efforts for what is \"routinely used in our laboratory\"; what is done is usually well done. It's just that …

Methods in Enzymology.

The sequence A–A–U–A–A–A is present in six different purified messenger RNA molecules (specifically the α- and β-globulin mRNAs of rabbit and human, the immunoglobulin light chain mRN A of mouse (MOPC 21) and the ovalbumin mRN A of chicken) about 20 residues away from the 3′-terminal poly(A) sequence. In addition, a large selection of the 3′ non-coding regions of rabbit and human globin mRN As (both the α- and β- globin mRN As) are 85% homologous, demonstrating that this region is significantly conserved in evolution.

3′ Non-coding region sequences in eukaryotic messenger RNA

An mRNA fraction coding for hexon polypeptide, the major virion structural protein, was purified by gel electrophoresis from extracts of adenovirus 2-infected cells late in the lytic cycle. The mRNA sequences in this fraction were mapped between 51.7 and 61.3 units on the genome by visualizing RNA-DNA hybrids in the electron microscope. When hybrids of hexon mRNA and single-stranded restriction endonuclease cleavage fragments of viral DNA were visualized in the electron microscope,branched forms were observed in which 160 nucleotides of RNA from the 5' terminus were not hydrogen bonded to the single-stranded DNA. DNA sequences complementary to the RNA sequences in each 5' tail were found by electron microscopy to be located at 17, 20, and 27 units on the same strand as that coding for the body of the hexon mRNA. Thus, four segments of viral RNA may be joined together during the synthesis of mature hexon mRNA. A model is presented for adenovirus late mRNA synthesis that involves multiple splicing during maturation of a larger precursor nuclear RNA.

https://www.pnas.org/content/pnas/74/8/3171.full.pdf

Spliced segments at the 5' terminus of adenovirus 2 late mRNA

Sites of transcription of polyadenylated and nonpolyadenylated RNAs for 10 human chromosomes were mapped at 5-base pair resolution in eight cell lines. Unannotated, nonpolyadenylated transcripts comprise the major proportion of the transcriptional output of the human genome. Of all transcribed sequences, 19.4, 43.7, and 36.9% were observed to be polyadenylated, nonpolyadenylated, and bimorphic, respectively. Half of all transcribed sequences are found only in the nucleus and for the most part are unannotated. Overall, the transcribed portions of the human genome are predominantly composed of interlaced networks of both poly A+ and poly A- annotated transcripts and unannotated transcripts of unknown function. This organization has important implications for interpreting genotype-phenotype associations, regulation of gene expression, and the definition of a gene.

http://science.sciencemag.org/content/sci/308/5725/1149.full.pdf

Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution

https://mmbr.asm.org/content/mmbr/47/1/1.full.pdf

Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles.

Polyadenylate sequences have been found covalently linked in heterogeneous DNA-like nuclear RNA of HeLa cells. This poly(A) material seems homogeneous in size and accounts for about 0.5% of such RNA. Similar poly(A) sequences were found in rapidly-labeled polyribosomal RNA, thought to be messenger RNA. A possible model for mRNA synthesis from large heterogeneous nuclear RNA precursor molecules is discussed.

https://www.pnas.org/content/pnas/68/6/1336.full.pdf

Polyadenylic Acid Sequences in the Heterogeneous Nuclear RNA and Rapidly-Labeled Polyribosomal RNA of HeLa Cells: Possible Evidence for a Precursor Relationship

The Isolation and Characterization of Adenosine Monophosphate-rich Polynucleotides Synthesized by Ehrlich Ascites Cells

Polynucleotide biosynthesis: formation of a sequence of adenylate units from adenosine triphosphate by an enzyme from thymus nuclei.

Biological polyadenylation, first recognized as an enzymatic activity, remained an orphan enzyme until poly A sequences were found on the 3′ ends of eukaryotic mRNAs. Their presence in bacteria viruses and later in archeae (ref. 338) established their universality. The lack of compelling evidence for a specific function limited attention to their cellular formation. Eventually the newer techniques of molecular biology and development of accurate nuclear processing extracts showed 3′ end formation to be a two-step process. Pre-mRNA was first cleaved endonucleolytically at a specific site that was followed by sequential addition of AMPs from ATP to the 3′ hydroxyl group at the end of mRNA. The site of cleavage was specified by a conserved hexanucleotide, AAUAAA, from 10 to 30 nt upstream of this 3′ end. Extensive purification of these two activities showed that more than 10 polypeptides were needed for mRNA 3′ end formation. Most of these were in complexes involved in the cleavage step. Two of the best characterized are CstF and CPSF, while two other remain partially purified but essential. Oddly, the specific proteins involved in phosphodiester bond hydrolysis have yet to be identified. The polyadenylation step occurs within the complex of poly A polymerase and poly A-binding protein, PABII, that controls poly A length. That the cleavage complex, CPSF, is also required for this step attests to a tight coupling of the two steps of 3′ and formation. The reaction reconstituted from these RNA-free purified factors correctly processes pre-mRNAs. Meaningful analysis of the role of poly A in mRNA metabolism or function was possible once quantities of these proteins most often over-expressed from cDNA clones became available. The large number needed for two simple reactions of an endonuclease, a polymerase and a sequence recognition factor, pointed to 3′ end formation as a regulated process. Polyadenylation itself had appeared to require regulation in cases where two poly A sites were alternatively processed to produce mRNA coding for two different proteins. The 64-KDa subunit of CstF is now known to be a regulator of poly A site choice between two sites in the immunoglobulin heavy chain of B cells. In resting cells the site used favors the mRNA for a membrane-bound protein. Upon differentiation to plasma cells, an upstream site is used the produce a secreted form of the heavy chain. Poly A site choice in the calcitonin pre-mRNA involves splicing factors at a pseudo splice site in an intron downstream of the active poly site that interacts with cleavage factors for most tissues. The molecular basis for choice of the alternate site in neuronal tissue is unknown. Proteins needed for mRNA 3′ end formation also participate in other RNA-processing reactions: cleavage factors bind to the C-terminal domain of RNA polymerase during transcription; splicing of 3′ terminal exons is stimulated by cleavage factors that bind to splicing factors at 3′ splice sites; nuclear export of mRNAs is linked to cleavage factors and requires the poly A II-binding protein. Most striking is the long-sought evidence for a role for poly A in translation in yeast where it provides the surface on which the poly A-binding protein assembles the factors needed for the initiation of translation. This adaptability of eukaryotic cells to use a sequence of low information content extends to bacteria where poly A serves as a site for assembly of an mRNA degradation complex in E. coli . Vaccinia virus creates mRNA poly A tails by a streamlined mechanism independent of cleavage that requires only two proteins that recognize unique poly A signals. Thus, in spite of 40 years of study of poly A sequences, this growing multiplicity of uses and even mechanisms of formation seem destined to continue.

Mary Edmonds

Papers

Polyadenylic Acid Sequences in the Heterogeneous Nuclear RNA and Rapidly-Labeled Polyribosomal RNA of HeLa Cells: Possible Evidence for a Precursor Relationship

The Isolation and Characterization of Adenosine Monophosphate-rich Polynucleotides Synthesized by Ehrlich Ascites Cells

Polynucleotide biosynthesis: formation of a sequence of adenylate units from adenosine triphosphate by an enzyme from thymus nuclei.

A history of poly A sequences: from formation to factors to function.

Polyadenylic Acid Sequences in Yeast Messenger Ribonucleic Acid