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What is in the structure? It is, of course, quality!
The determination of the three-dimensional (3-D) structure of DNA in 1953 heralded the beginning of molecular biology. At the same time, we saw one of the first examples of how the 3-D structure of a biomolecule reveals its function. The 3-D structure of DNA immediately suggested how the genetic information is passed to the progeny. Eventually, the discovery of DNA structure led to the understanding of how genetic information accounts for the final product, which is protein synthesis. For the past 40 years, research in molecular biology has led to the identification of a cascade in gene regulation from its packing into chromosomes to transcription, splicing, modifications, protein synthesis, and, finally, the death of proteins. Eventually, knowledge of the mechanisms involved in these events led to manipulation of genes, recombinant DNA, and cloning technology, all of which helped us grasp the function of genes and their role in the study of differentiation, development, and diseases.
As the major players at all the different levels of gene regulation were discovered, it became apparent that the final mechanisms will be best revealed when we can observe the action of enzymes and genes at the 3-D level. Information gathered by biochemical and molecular experiments could identify the function of an enzyme, say the role of DNA polymerase in replication or the role of helicase in unwinding DNA.
PRIMER Transcription produces an RNA transcript, which contains the entire nucleotide sequence as it is imprinted in the DNA. In other words, the primary RNA transcript also contains the intronic sequences. These intronic sequences must be removed to create a continuous coding sequence that will be able to be translated to a protein. The process of intron removal is called splicing. However, not all introns are spliced-out the same way. Depending on the mechanism involved in their splicing, the introns are divided into three groups. In this chapter, I will present the steps of intron removal, emphasizing the 3-D structure of RNAs and proteins involved. Some introns are removed with the help of proteins, but others self-catalyze their cleavage. Therefore, the 3-D structure of the introns must be important in splicing and will be presented in order to visualize the mechanism. The determination of the 3-D structure of self-catalyzed introns is another celebrated example of how the structure reveals the function.
Transcription results in the production of the primary RNA transcript that is a copy of the DNA sequences. These sequences also contain the introns that do not contribute to the translation process that decodes the DNA sequences and produce proteins. Therefore, the intronic sequences must be removed. This process of splicing is very complex and depends on the type of introns. Some introns require a complex machinery, composed of proteins and RNAs, called the spiceosome.
PRIMER As the polypeptide is born and exits from the ribosome, it is bound by factors that will enable it to pass through the membranes of the endoplasmic reticulum. This is possible because all secreted proteins contain a signal in the very N-terminus, the signal peptide. This is recognized by the signal recognition particle that brings it to the receptor in the membrane and facilitates the passing. Then, the polypetide has to fold into its unique 3-D structure. We already know how important this is. The function of a protein depends on its 3-D structure. The folding must be correct; otherwise, the protein will not be allowed to perform its tasks. Folding involves many factors, called chaperones. The 3-D structures of chaperones also provide stunning images of the mechanism of folding. Alas, nothing lives forever! And this rule applies to proteins as well. In fact, it is central to regulation that the protein will die after some time. If a protein does not die, it will accumulate and induce diseases. Eventually, aged proteins will be recognized and brought to their demise with the help of particular proteins and structures. These processes are examined and compared in prokaryotes and eukaryotes. It will be the end of the protein, the end of our journey, and the end of the book.
The synthesis of the polypetide is not the end of the story. The protein is not ready for work yet. Several modifications must take place.
PRIMER When the mRNA is in the cytoplasm, it is recognized by factors and complexed with the ribosome, which is the place where the message is decoded and the protein is built up. We are rather fortunate because most of the players participating in protein synthesis are known at the 3-D level; thus, we can reconstruct all the steps of protein synthesis at the 3-D level. Some of the figures, especially the ones depicting the process of protein synthesis with the ribosome, are so stunning that I virtually feel I am there viewing the process first hand. For this spectacle to flow in the most comprehensive way possible, I selected a certain sequence for the presentation. First, I present the 3-D structure of the two major players in protein synthesis: tRNA (alone and complexed with aatRNA synthetase) and ribosome. The reader must become very familiar with both, especially with the ribosome. Even though we know the structure of the ribosome in high resolution, I first introduce its structure by showing a series of images from low to high resolution. This approach should enable the reader to understand the structure better. Having introduced the basic players, I proceed with the first stage of protein synthesis, the initiation. This is compared in both prokaryotes and eukaryotes, and the known 3-D structure of initiation factors and their interaction with rRNA are presented.
PRIMER Unwinding of DNA is not only restricted during replication but also occurs during transcription, even though the unwinding takes place in a different way. Transcription is a very important step, perhaps the most important step in regulation. Transcription determines which genes will be expressed and, therefore, proceed for decoding and protein synthesis. During the process of transcription, the 3′ → 5′ noncoding DNA strand becomes the template for the synthesis of RNA. The RNA transcript, therefore, keeps the sequence order of the coding strand, 5′ → 3′. The main enzyme here is RNA polymerase. Regulation at this level is very unique and markedly different in prokaryotes and eukaryotes. Therefore, for clarity, I describe transcription in prokaryotes first (this chapter) and continue with transcription in eukaryotes in Chapter 6).
The first part of this chapter examines the structure of the prokaryotic RNA polymerase and the mechanisms of transcription initiation and elongation as revealed by 3-D complexes of RNA polymerase and DNA. In the second part, I present some of the mechanisms involved in regulating gene transcription in prokaryotes. Having as a focus to present these events at the 3-D level, my aim is not to be redundant, and in doing so I have omitted information that can be found in microbiology and biochemistry books. Therefore, the basic structural motifs involved in transcriptional regulation are presented in a few regulators to pinpoint similarities and differences.
PRIMER The three-dimensional structure of nucleic acids and proteins as it pertains to the mechanisms involved in gene regulation is the major focus of this book. Therefore, the reader will encounter many 3-D structures. The first chapter of the book presents the very basic ideas behind the three-dimensional aspects of biomolecules. The first part deals with the techniques used to determine 3-D structures. The presentation is virtually for the layperson. Then the basic structural elements found in proteins are examined. Having done this, we examine a particular 3-D structure (that includes both DNA and protein) presented with different modeling. This exercise will help you to become familiar with the different ways that scientists present their 3-D structures. We use different models because one aspect of structure and function can be better represented with one model, whereas another aspect is more suited to a different model.
This book deals with the three-dimensional aspects of gene regulation. The reader will encounter numerous three-dimensional structures, but this should not scare anybody away. Unfamiliar readers might think that interpreting these structures is difficult, but this is not true. All we need is a basic introduction into the three-dimensional aspects of proteins and nucleic acids and the way that it can be represented. The basic 3-D structure of a protein can be reduced to two elements: the alpha helix and the beta strand (and loops that connect them). The complicated 3-D structure of a protein is a combination of several of these elements.
PRIMER DNA is not free in the nucleus; it is bound by the proteins that package it. This fact is important because DNA must be accommodated in a small place. Also, as we will see in Chapter 6, the packaging provides a level of transcriptional control. In this chapter, the goal is to become familiar with the proteins that are involved in packaging and their effects on DNA. The first part of the chapter examines the 3-D structure of the nucleosome and its components, the histones. This structure is the highest order of DNA packaging. Other proteins (nonhistone), however, have the ability to bend DNA and, therefore, provide another degree of organization. Such architectural proteins are important because linear DNA is not very efficient when it comes to regulation. Regulatory elements are often separated by long distances, and they must come close for interaction. Also, bending DNA makes it more accessible for interactions with proteins. These nonhistone proteins possess different structural motifs that mediate interaction with DNA. Their structural characteristics and effects on DNA are presented. The importance of these architectural proteins is also stressed in later chapters.
PACKING DNA IN THE CHROMOSOMES
The genetic material, DNA, is packaged into chromosomes as chromatin, which is the DNA and chromosomal proteins. The way that this packaging is achieved is suitable for DNA accommodation in the nucleus. It also most likely plays a role in DNA regulation. The packing of DNA is characterized by coils, loops, and coils within the loops.
PRIMER The spliced and modified mRNA must be transported to the cytoplasm for translation that will result in protein synthesis, which is the ultimate goal of transcription. The question addressed in this chapter involves the degree to which the topology of transcription and movement of the transcripts toward the nuclear pores is organized. This area of inquiry is a relatively new, and experiments are possible only by using modern image analysis techniques. Evidence is presented that transcription and movement of transcripts are organized topologically in the nucleus. Such an organization is compared with DNA replication. This information is very useful in understanding the spatial employment of these events.
So far, we have covered areas from the organization of DNA in the chromosomes to replication and the different steps of transcription. We have done all this with the three-dimensional aspects of molecular biology in mind; therefore, our study was oriented toward precise and organized structures. We have seen how precisely the splicing apparatus is folded to create unique structures and arrangement for the removal of the introns. Such structures are correlated to functions and also have an evolutionary history to account for them. What is, however, the picture when we move ourselves from the small scales to the larger ones? Are all the centers of transcription organized? Are all the splicing centers organized? Is their location in the nucleus determined or unique?
Results from a QTL experiment on growth and carcass traits in an experimental F2 cross between Iberian and Landrace pigs are reported. Phenotypic data for growth, length of carcass and muscle mass, fat deposition and carcass composition traits from 321 individuals corresponding to 58 families were recorded. Animals were genotyped for 92 markers covering the 18 porcine autosomes (SSC). The results from the genomic scan show genomewide significant QTL in SSC2 (longissimus muscle area and backfat thickness), SSC4 (length of carcass, backfat thickness, loin, shoulder and belly bacon weights) and SSC6 (longissimus muscle area, backfat thickness, loin, shoulder and belly bacon weights). Suggestive QTL were also found on SSC1, SSC5, SSC7, SSC8, SSC9, SSC13, SCC14, SSC16 and SSC17. A bidimensional genomic scan every 10 cM was performed to detect interaction between QTL. The joint action of two suggestive QTL in SSC2 and SSC17 led to a genome-wide significant effect in live weight. The results of the bidimensional genomic scan showed that the genetic architecture was mainly additive or the experimental set-up did not have enough power to detect epistatic interactions.
We investigate the joint effects of gene flow and selfing on the level of inbreeding depression, heterosis and genetic load in a subdivided population at equilibrium. Low gene flow reduces inbreeding depression and substantially increases heterosis. However, in highly self-fertilizing populations, inbreeding depression is independent of the amount of gene flow. When migration occurs via pollen, consanguinity of the reproductive system could have a negative influence on subpopulation persistence, in contrast to the case of isolated populations. However, with only seed migration, genetic load and heterosis depend mildly on the mating system. From an evolutionary point of view, we reach two main conclusions: first, outcrossing is selected for if gene flow is low; second, intermediate levels of gene flow could promote mixed mating systems, especially when migration occurs through pollen.
Two molecular maps of Triticum monococcum L were produced and integrated. The integrated map includes a total of 477 markers, 32 RFLPs, 438 AFLPs, one morphological (soft glume (Sog)) and six storage-protein markers, and covers 856 cM. The trait Sog with the recessive allele sog maps to linkage group 2S. Probably, this is the T. monococcum homologue of Tg and Tg2 in hexaploid and tetraploid wheats, respectively. Loci coding for seed storage proteins were allocated to chromosomes 1L (HMW GLU1,2 and Glu1), 1S (LMW GLU6,7, LMW GLU1-4, ωGLI1-4, γGLI5 and Gli-1) and 6L (α/βGLI7-14). Parameters related to bread-making quality (SDS sedimentation volume, specific sedimentation volume (SSV) and total protein content) were studied in one of the two populations. A QTL that is consistently present across environments was detected for SDS sedimentation volume and for SSV. The position of the QTL on chromosome 1S was in close agreement with the map positions of storage-protein loci. A second QTL was mapped on chromosome 5. For protein content, two significant QTLs were mapped to linkage groups 1 and 5.
DNA variation was studied in a 2.2 kb region of the regulatory gene Atmyb2 using 20 ecotypes of Arabidopsis thaliana and one accession each of Arabis gemmifera and Arabidopsis himalaica. Nucleotide diversity (π) in the region was 0.0027, which was lower than for other loci in A. thaliana. The MYB domain of the Atmyb2 gene (π = 0.0036) had a larger variation than the non-MYB region (π = 0.0013). Tajima's test and Fu and Li's test did not give a significant result. In contrast to the low level of polymorphism, the degree of divergence of the Atmyb2 region was higher between A. thaliana and A. gemmifera (K = 0.0730) than for other loci. The MYB domain (K = 0.0436) had smaller divergence than the non-MYB region (K = 0.0939). The HKA test detected significant discordance in the ratio of polymorphism to divergence in some comparisons. The pattern of low polymorphism and high divergence, which is mainly observed in the non-MYB region of the gene, is inconsistent with the neutral mutation theory. Strong purifying selection after establishment of A. thaliana and a species-specific adaptive process could be invoked to account for this pattern of polymorphism and divergence of Atmyb2.
In the first 25 generations of his classical mutation accumulation experiment, T. Mukai estimated a large rate of early linear decay for the relative viability of Drosophila melanogaster chromosome II (ΔMII = 0.004). Mukai forced through zero the regression of viability decline on generation number, but it has recently been shown (Fry, 2001) that a similar decline (ΔMII = 0.006) is obtained from unforced regression even if generation 32 instead of generation 25 (whose validity has been questioned) is included. We show that, from the perspective of the whole long-term experiment, it is hard to decide up to which generation viability can be considered to decline linearly. Depending on this decision, and on whether or not the regression is forced through the origin, very different estimates are obtained. Furthermore, the particular behaviour of the lines used as control suggests that they could have been different from the remaining lines at the beginning of the experiment, and casts doubts on the adequacy of a forced regression. Estimates from the linear unforced regression (ΔMII = 0.011) or from the linear term in a quadratic unforced regression (ΔMII = 0.001) are very different. The data fit both models very well, and the choice between them should be based on biological grounds.
An unusual chromosomal hybrid zone of the house mouse, Mus musculus domesticus, exists in Upper Valtellina, Northern Italy, consisting of four Robertsonian (Rb) races and the standard (all-acrocentric, or 2n = 40) race, all hybridizing freely within 10 km2. The hybrid zone in Valtellina provides an excellent opportunity to study the role of Rb fusions in reproductive isolation and speciation. This hybrid zone has already been well studied for the distribution of Rb fusions and the fertility of hybrids, but in order to understand the dynamics of the zone, a basic understanding of the origin and genetic similarity of the chromosomal races is necessary. This paper presents the results of three different methods of measuring genetic differentiation: multivariate analysis of morphological traits and analyses of allozyme variation and mitochondrial DNA sequences. The standard race is clearly distinguishable from the three Rb races by all three methods, but the Rb races are not distinguishable from one another. This provides strong evidence for our previous suggestions that the well-established Rb races in Valtellina are closely related, and that the standard race was introduced into the valley more recently from a distant source. The fact that the Rb races are indistinguishable is also consistent with our hypothesis that a within-village speciation event involving two of the races (Hauffe & Searle, 1992) was a recent occurrence. The low level of allozyme heterozygosity among the Rb races suggests that these populations are the products of at least one bottleneck. The present article substantially extends earlier studies and provides the first detailed morphological and molecular analysis of this complex hybrid zone.
In Drosophila melanogaster, the maternally inherited endocellular microbe Wolbachia causes cytoplasmic incompatibility (CI) in crosses between infected males and uninfected females. CI results in a reduction in the number of eggs that hatch. The level of CI expression in this species has been reported as varying from partial (a few eggs fail to hatch) to nonexistent (all eggs hatch). We show that male age in this host species has a large impact on the level of CI exhibited and explains much of this variability. Strong CI is apparent when young males are used in crosses. CI declines rapidly with male age, particularly when males are repeatedly mated. Wolbachia from a Canton S line that was previously reported as not causing CI does in fact induce CI when young males are used in crosses, albeit at a weaker level than in other D. melanogaster strains. The strain differences in CI expression are due to host background effects rather than differences in Wolbachia strains. These results highlight the importance of undertaking crosses with a range of male ages and nuclear backgrounds before ascribing particular host phenotypes to Wolbachia strains.
Dissection of quantitative traits to the nucleotide level requires phenotypic and genotypic analysis of traits on a genome scale. Here we discuss the set of community-wide genetic and molecular resources, including panels of specific types of inbred lines and high density resequencing and SNP detection, that will facilitate such studies.