Replication

There is no more critical issue in the origin of life than a method for the faithful and timely replication of genetic information. Genes are encoded in deoxyribonucleic acid (DNA), which is made of four types of nucleotides , distinguished by the bases adenine (A), guanine (G), cytosine (C), and thymine (T). When James Watson and Francis Crick discovered the double helical structure of deoxyribonucleic acid (DNA), they also recognized that DNA replication could occur by opening the double helix into single strands, and from those templates creating new complementary strands by the principle of base pairing . As an overview, their proposal was correct. The details of the chemistry, of the enzymes involved, and of the structure on which replication occurs tell a fascinating story that is far more complex than Watson and Crick anticipated.

Semiconservative Replication

Chromosomes are the extended molecules of DNA that carry genes in both bacteria and eukaryotes. Bacterial chromosomes are usually circular, with the double helix looping around to make a complete circle. Eukaryotic chromosomes are linear, with the double helix sealing up at the two distant ends. In both cases, the result of replication is that one double helix with its two complementary strands of nucleotides becomes two identical double helices with the same sequence of nucleotides. In this way, the genetic material of a cell is passed along unchanged through all the descendants of the original cell (except for replication errors or other mutations).

Each new helix includes one original side and one new side, and so the replication process is said to be "semiconservative." Semiconservative replication was discovered by Matthew Meselson and Frank Stahl, who grew bacteria with radioactive nucleotides. Fully radioactive chromosomes became half as radioactive after one round of replication, indicating that half the original chromosome was preserved in each new copy.

Replication Forks and Accessory Enzymes

Replication is a huge task, whether in bacteria or in eukaryotes. There are several physical and biochemical challenges the cell must overcome. First, the site or sites at which to begin replication must be located and the proper enzymes collected there. Second, the double helix must be unwound to expose the two strands. This imposes twisting strain on the portions of the helix farther away from the unwinding site, much like untangling a twisted phone cord does, and those forces must be relieved to prevent breakage of the DNA strands. Complementary nucleotides must be put in place and linked to form a new strand, and errors must be checked and corrected.

The orientation of the two strands poses an additional challenge. Because of the way the deoxyribose sugar is structured, the sugar, and hence the whole DNA strand, has a direction, an up versus down, so to speak. The two sides are oriented with up and down directions opposite, in a so-called antiparallel fashion. In biochemical terms, one direction is 5′-3′ ("five-prime to three-prime"), while the other side is oriented 3′-5′ . The consequence of this arises because the enzymes that perform replication only function in one direction. The solution to this problem is discussed below.

Much of the understanding of DNA replication has come from studying the bacterium Escherichia coli. In bacterial chromosomes one site, called the origin of replication, has a sequence of base pairs to which an initiator protein binds. The initiator protein attracts enzymes called helicases that interrupt base pairing in a way that separates the double helix into a short region of two single strands. Other binding proteins then attach to maintain the single strand separation on the double-stranded region. Adjacent to the single-stranded region, an enzyme called gyrase cleaves and reforms the sugar-phosphate backbones in the double strand helix, which relieves the strain that the single strand separation causes. Separating the double-stranded helix into a region of two single strands creates a "replication bubble" initially of a few hundred nucleotides. Within the replication bubble, each strand serves as the template for a new chain; these chains grow in opposite directions because the templates have opposite 5′ to 3′ polarity. As the chains elongate, the replication bubble expands in both directions, and the ends become known as replication forks. As replication occurs, the helicase molecules are pushed toward the fork where the double strand separates into single strands, thus moving the fork and extending the region of single strands.

Priming and Elongation

E. coli use three enzymes for replications, called pol I, pol II, and pol III. Early work with pol I showed that replication adds a free nucleotide to the 3′ OH group of the last nucleotide in the growing chain (see Figure 1). Each nucleotide-added hydrogen bonds to the complementary nucleotide in the template single strand. The 3′ OH of the last nucleotide attacks the high-energy triphosphate group at the 5′ position of the free nucleotide, splitting off two phosphates and forming a covalent bond to the innermost phosphate. This binds the new nucleotide to the existing chain.

Some anticancer and antiviral drugs are nucleotides missing the 3′ OH. Such "dideoxy" nucleotides shut down replication after being incorporated into the strand. Fast-replicating DNA in cancer cells or viruses is inactivated by these drugs.

To begin elongation, DNA polymerases require an existing chain with a 3′ OH end. This posed the problem of how replication could ever begin, since the needed 3′ OH is on the newly replicated strand. The key to understanding the initiation of replication came with the discovery of ribonucleic acid (RNA) priming, which does not require an existing 3′ OH to start the process. In priming, an enzyme called primase places a short sequence of RNA nucleotides into position at the origin of replication. This sequence is complementary to the 3′ end of the single-stranded portion of the template at that point. DNA polymerase then adds nucleotides to the RNA's 3′ OH, continuing replication, until they reach the end of the complementary template.

Since polymerase only elongates in a 5′ to 3′ direction, at each replication fork only one chain is elongating in a smooth, unbroken fashion. The continuous elongation product is termed the "leading strand," while the discontinuous product is the "lagging strand." For every new section on the lagging strand, primase creates a new RNA primer and 3′ OH end to start DNA chain elongation. Called Okazaki fragments after their discoverer, each short fragment lasts only a brief period before the primer ribonucleotides are digested and replaced with DNA nucleotides, which are then ligated ("tied") to the adjacent nucleotides by DNA ligase. Ligation of the Okazaki fragments into long chains completes synthesis of the lagging strand (see Figure 2). The pol III enzyme is a large complex of subunits that catalyzes both leading-strand and lagging-strand elongation and has roles in proofreading and replacing mismatched nucleotides. The pol I enzyme digests the RNA nucleotides and replaces them with DNA nucleotides; it also proofreads and replaces incorrect with correct nucleotides. It is thought that pol II mostly repairs replication errors.

Replication in the E. coli chromosome is bidirectional and continues in opposite directions until the two replication forks meet about halfway around the circular chromosome. Replication is then complete.

Special Features of Eukaryotic Replication

Replication of eukaryotic chromosomes is more complex inasmuch as they are linear (versus circular) and usually much larger than bacterial chromosomes. DNA replication is restricted to the S, or synthesis, phase during the cell cycle , between mitotic divisions. As a result of replication, each chromosome consists of two identical chromatids joined together, which are then separated into daughter cells by mitotic division.

Instead of a single origin of replication, as in bacteria, eukaryotic chromosomes have many origins for each chromosome in keeping with their much larger size. Replication proceeds bidirectionally from each origin until it meets the replication fork from the adjacent origin. A replicon refers to the interval replicated from one origin. This concept is shaky, however, since there is evidence that origins of replication are somewhat specific to the stage and tissue the cells are in, rather than being a permanent physical property. For example, nuclei in the Drosophila embryo divide every nine minutes initially. This requires the fastest replication of chromosomes known and utilizes many origins. Later stages use fewer origins.

Eukaryotes have five polymerases, termed alpha, beta, gamma, delta, and epsilon. Replication of nuclear DNA utilizes the alpha and delta polymerases. Alpha polymerase is a complex of several subunits, one of which has primase activity when it is in the complex. The alpha polymerase is thought to carry out synthesis of the lagging strand, whereas the delta polymerase, also a complex of subunits but lacking primase activity, carries out synthesis of the leading strand. As in the prokaryotes , helicase and gyrase are required to unwind the double helix ahead of the replication fork. The alpha and delta polymerases function in proofreading and correction as well. The beta and epsilon polymerases are thought to carry out nuclear DNA repair. The gamma polymerase replicates the mitochondrial genome . It lacks the error correction mechanism of the other polymerases, with the result that the mutation rate in mitochondrial replication is substantially higher than it is in replication of nuclear DNA.

Telomeres and Telomerase

Because eukaryotic chromosomes are linear and have ends, another enzyme, called telomerase, is necessary but is not found in the prokaryotes. The problem with chromosome ends, called telomeres, is that the 3′ template for the lagging strand cannot be primed at the last nucleotides because there is no further DNA on which to build. By itself, this would reduce the length of the lagging strand and the chromosome would get shorter at each replication. Telomeric DNAs, which are repeats of a specific sequence of six nucleotides, are normally present at the ends of chromosomal DNA and avoid this problem. Telomerase is a hybrid protein-RNA molecule; the RNA sequence is complementary to several repeat lengths of the telomeric DNA. The telomerase uses the RNA sequence to bind to the template end of telomeric DNA and uses the overhang protein portion to add DNA nucleotides to the template, extending it beyond its normal length. With several movements of the enzyme outward and reiterations of this process, the template 3′ end is extended sufficiently to allow DNA polymerase to complete synthesis of a normal length lagging strand. In multicellular organisms, somatic cells usually cease mitotic division during development and lack telomerase activity thereafter. Cancer cells abnormally turn their telomerase back on, which enables the cell to divide continually. Telomerase is a target of drug research for the combat of cancer.

see also Bacterial Cell; Cell Cycle; Chromosome, Eukaryotic; DNA; Nucleotides; RNA; Transcription

John Merriam

Bibliography

Cotterill, Sue, ed. Eukaryotic DNA Replication: A Practical Approach. Oxford: Oxford University Press, 1999.

Kornberg, Arthur, and Tania Baker. DNA Replication. New York: W. H. Freeman and Company, 1991.

Replication

DNA is the carrier of genetic information. Before a cell divides, DNA must be precisely copied, or "replicated," so that each of the two daughter cells can inherit a complete genome, the full set of genes present in the organism. In eukaryotes , the DNA molecules that make up the genome are packaged with proteins into chromosomes, each of which contains a single linear DNA molecule. Eukaryotic chromosomes are found in a special compartment called the cell nucleus. The genomes of bacterial cells (prokaryotes ), which lack a nucleus, are typically circular DNA molecules that associate with special structures in the cell membrane. Despite the hundreds of millions of years of evolutionary history separating eukaryotes and prokaryotes, the features of the replication process have been highly conserved between them.

Overview

The DNAs that make up the genomes of bacteria and eukaryotic cells are double-stranded molecules in which each strand is composed of subunits called nucleotides. DNA nucleotides have a direction, in the same way that an arrow has a head and a tail. In DNA strands, the head is the 3′ ("three prime") end of the strand, and the tail is the 5′ ("five prime") end. As a result, each strand also has a direction, whose ends are referred to as the 3′ and 5′ ends. The two strands of DNA run in opposite directions, and are wound around each other in a double helix, with the strands held together by hydrogen bonds between paired bases of the nucleotides (A pairs with T, and G pairs with C).

During the process of DNA replication, the strands are unwound by an enzyme called DNA helicase, and a new strand of DNA is synthesized on each of the old (template ) strands by an enzyme called DNA polymerase, which joins incoming nucleotides together in a sequence that is determined by the sequence of nucleotides present in the template strand. DNA replication is said to be semiconservative because each of the two identical daughter molecules contains one of the two parental template strands paired with a new strand. Prokaryotic replication can take as little as twenty minutes, while replication in eukaryotes takes considerably longer, approximately eight hours in mammals.

Initiation of DNA Replication

DNA replication begins (initiates) at special sites called origins of DNA replication. Eukaryotic DNAs each contain multiple replication origins, spaced at intervals of approximately 100,000 base pairs (100 kilobase pairs, or 100 kb) along the length of the DNA. There are 6 billion base pairs in the human genome, located on forty-six chromosomes, and so each chromosome will have many origins of replication. Prokaryotic chromosomes typically have a single replication origin.

Replication origins are composed of special sequences of DNA that are recognized by replication initiator proteins, which bind to the origin sequences and then help to assemble other proteins required for DNA replication at these sites. The eukaryotic replication initiator protein is a complex containing six different subunits called the origin recognition complex (ORC). The bacterial replication initiator protein is called the dna A protein. The timing of DNA replication is regulated by controlling the assembly of complexes at replication origins.

The distinct steps in the initiation of replication are understood better in bacteria than in eukaryotes, but several key steps are common to both. The first step is a change in the conformation of the initiator protein, which causes limited "melting" (that is, the separation of the two strands) of the double-stranded DNA next to the initiator binding site, thus exposing single-stranded regions of the template (Figure 1). Two more proteins, DNA helicase and DNA primase, then join the complex. Replication initiation is triggered by the activation of the helicase and primase, and the subsequent recruitment of DNA polymerase. In prokaryotes, the particular form of the enzyme is called DNA polymerase III. Other proteins are also recruited, each of whose functions are discussed below.

The Replication Fork

The separation of the two template strands and the synthesis of new daughter DNA molecules creates a moving "replication fork" (Figure 2), in which, double-stranded DNA is continually unwound and copied. The unwinding of DNA poses special problems, which can be visualized by imagining pulling apart two pieces of string that are tightly wound around each other. The pulling apart requires energy; the strands tend to rewind if not held apart; and the region ahead of the separated strands becomes even more tightly twisted.

Proteins at the replication fork address each of these problems. DNA polymerases are not able to unwind double-stranded DNA, which requires energy to break the hydrogen bonds between the bases that hold the strands together. This task is accomplished by the enzyme DNA helicase, which uses the energy in ATP to unwind the template DNA at the replication fork. The single strands are then bound by a single-strand binding protein (called SSB in bacteria and RPA in eukaryotes), which prevents the strands from reassociating to form double-stranded DNA. Unwinding the DNA at the replication fork causes the DNA ahead of the fork to rotate and become twisted on itself. To prevent this from happening, an enzyme called DNA gyrase (in bacteria) or topoisomerase (in eukaryotes) moves ahead of the replication fork, breaking, swiveling, and rejoining the double helix to relieve the strain.

Leading Strands and Lagging Strands

The coordinated synthesis of the two daughter strands posed an important problem in DNA replication. The two parental strands of DNA run in opposite directions, one from the 5′ to the 3′ end, and the other from the 3′ to the 5′ end. However, all known DNA polymerases catalyze DNA synthesis in only one direction, from the 5′ to the 3′ end, adding nucleotides only to the 3′ end of the growing chain. The daughter strands, if they were both synthesized continuously, would have to be synthesized in opposite directions, but this is known not to occur. How, then, can the other strand be synthesized?

The resolution of the problem was provided by the demonstration that only one of the two daughter strands, called the leading strand, is synthesized continuously in the overall direction of fork movement, from the 5′ to the 3′ end (see Figure 3). The second daughter strand, called the lagging strand, is made discontinuously in small segments, called Okazaki fragments in honor of their discoverer. Each Okazaki fragment is made in the 5′ to 3′ direction, by a DNA polymerase whose direction of synthesis is backwards compared to the overall direction of fork movement. These fragments are then joined together by an enzyme called DNA ligase.

The Need for Primers

Another property of DNA polymerase poses a second problem in understanding replication. DNA polymerases are unable to initiate synthesis of a new DNA strand from scratch; they can only add nucleotides to the 3′ end of an existing strand, which can be either DNA or RNA. Thus, the synthesis of each strand must be started (primed) by some other enzyme.

The priming problem is solved by a specialized RNA polymerase, called DNA primase, which synthesizes a short (3 to 10 nucleotides) RNA primer strand that DNA polymerase extends. On the leading strand, only one small primer is required at the very beginning. On the lagging strand, however, each Okazaki fragment requires a separate primer.

Before Okazaki fragments can be linked together to form a continuous lagging strand, the RNA primers must be removed and replaced with DNA. In bacteria, this processing is accomplished by the combined action of RNase H and DNA polymerase I. RNase H is a ribonuclease that degrades RNA molecules in RNA/DNA double helices. In addition to its polymerase activity, DNA polymerase I is a 5′-to-3′ nuclease, so it too can degrade RNA primers. After the RNA primer is removed and the gap is filled in with the correct DNA, DNA ligase seals the nick between the two Okazaki fragments, making a continuous lagging strand.

DNA Polymerase

The two molecules of DNA polymerase used for the synthesis of both leading and lagging strands in bacteria are both DNA polymerase III. They are actually tethered together at the fork by one of the subunits of the protein, keeping their progress tightly coordinated. Many of the other players involved are also linked, so that the entire complex functions as a large molecular replicating machine.

DNA polymerase III has several special properties that make it suitable for its job. Replication of the leading strand of a bacterial chromosome requires the synthesis of a DNA strand several million bases in length. To prevent the DNA polymerase from "falling off" the template strand during this process, the polymerase has a ring-shaped clamp that encircles and slides along the DNA strand that is being replicated, holding the polymerase in place. This sliding clamp has to be opened like a bracelet in order to be loaded onto the DNA, and the polymerase also contains a special clamp loader that does this job.

A second important property of DNA polymerase III is that it is highly accurate. Any mistakes made in incorporating individual nucleotides cause mutations , which are changes in the DNA sequence. These mutations can be harmful to the organism. The accuracy of the DNA polymerase results both from its ability to select the correct nucleotide to incorporate, and from its ability to "proofread" its work.

Appropriate nucleotide selection depends on base-pairing of the incoming nucleotide with the template strand. At this step, the polymerase makes about one mistake per 1,000 to 10,000 incorporations. Following incorporation, the DNA polymerase has a way of checking to see that the nucleotide pairs with the template strand appropriately (that is, A only pairs with T, C only pairs with G). In the event that it does not, the DNA polymerase has a second enzymatic activity, called a proofreading exonuclease, or a 3′-to-5′ exonuclease, that allows it to back up and remove the incorrectly incorporated nucleotide. This ability to proofread reduces the overall error rate to about one error in a million nucleotides incorporated. Other mechanisms detect and remove mismatched base pairs that remain after proofreading and reduce the overall error rate to about one error in a billion.

Features of Replication in Eukaryotic Cells

The steps in DNA replication in eukaryotic cells are very much the same as the steps in bacterial replication discussed above. The differences in bacterial and eukaryotic replication relate to the details of the proteins that function in each step. Although amino acid sequences of eukaryotic and prokaryotic replication proteins have diverged through evolution, their structures and functions are highly conserved. However, the eukaryotic systems are often somewhat more complicated.

For example, bacteria require only a single DNA polymerase, using DNA polymerase III for both leading and lagging strand synthesis, and are able to survive without DNA polymerase I. In contrast, eukaryotes require at least four DNA polymerases, DNA polymerases α, δ, ε, and σ. DNA polymerases δ and ε both interact with the sliding clamp, and some evidence suggests that one of these polymerases is used for the leading strand and the other for the lagging strand. One required function of DNA polymerase σ is the synthesis of the RNA primers for DNA synthesis. The precise role of DNA polymerase is not yet known. A second example is removal of the RNA primers on Okazaki fragments. In eukaryotes, primer removal is carried out by RNase H and two other proteins, Fen1 and Dna2, which replace the 5′-to-3′ exonuclease provided by the bacterial DNA polymerase I in bacteria.

Replication continues until two approaching forks meet. The tips of linear eukaryotic chromosomes, called telomeres , require special replication events. Bacterial chromosomes, which contain circular DNA molecules, do not require these special events.

Regulating Replication

DNA replication must be tightly coordinated with cell division, so that extra copies of chromosomes are not created and each daughter cell receives exactly the right number of each chromosome. DNA replication is regulated by

controlling the assembly of complexes at replication origins. In bacteria, the accumulation of the initiator protein, dnaA, seems to be an important factor in determining when replication begins.

In eukaryotes, DNA replication and cell division are separated by two "gap" cell cycle phases (G₁ and G₂), during which neither DNA replication nor nuclear division occurs. DNA replication occurs during the S (or synthesis) phase, but ORC is thought to bind replication origins throughout the cell cycle. During the G₁ phase of the cell cycle, ORC helps to assemble other replication initiation factors at replication origins to make so-called pre-replicative-complexes (pre-RCs) that are competent to initiate replication during S phase. These other initiation factors include a protein called Cdc6 and a family of six related MCM ("mini-chromosome maintenance") proteins. The functions of these proteins are not yet known; however, the MCM proteins are currently the best candidate for the eukaryotic replicative helicase, and Cdc6 is necessary for MCM proteins to bind DNA. DNA polymerase also assembles on origins during this time.

Replication initiation is actually triggered at the beginning of S phase by the phosphorylation (addition of a phosphate group to) of one or more proteins in the pre-RC. The enzymes that phosphorylate proteins in the pre-RC are called protein kinases. Once they become active, they not only trigger replication initiation, but they also prevent the assembly of new pre-RCs. Therefore, replication cannot begin again until cells have completed cell division and entered G₁ phase again.

see also Cell Cycle; Chromosome, Eukaryotic; Chromosome, Prokaryotic; DNA; DNA Polymerases; DNA Repair; Mutation; Nucleases; Nucleotide; Nucleus; Telomere.

Carol S. Newlon

Bibliography

Baker, T. A., and S. P. Bell. "Polymerases and the Replisome: Machines within Machines." Cell 92 (1998): 295-305.

Cooper, Geoffrey M. The Cell: A Molecular Approach. Washington, DC: ASM Press, 1997.

Herendeen, D. R., and T. J. Kelly. "DNA Polymerase III: Running Rings Around the Fork." Cell 84 (1996): 5-8.

Lodish, Harvey, et al. Molecular Cell Biology, 4th ed. New York: W. H. Freeman, 2000.

Stillman, B. "Cell Cycle Control of DNA Replication." Science 274 (1996): 1659-1664.

Internet Resource

Davey, M., and M. O'Donnell. "DNA Replication." Genome Knowledge Base Website. <http://gkb.cshl.org/db/index>.

Inhibitors of viral helicaseprimase enzymes are being tested as a new treatment for herpes virus infection.

Chromosome

A chromosome is a threadlike structure found in the nucleus of most cells. It carries genetic material in the form of a linear sequence of deoxyribonucleic acid (DNA ). In prokaryotes, or cells without a nucleus, the chromosome represents circular DNA containing the entire genome. In eukaryotes, or cells with a distinct nucleus, chromosomes are much more complex in structure. The function of chromosomes is to package the extremely long DNA sequence. A single chromosome (uncoiled) could be as long as three inches and therefore visible to the naked eye. If DNA were not coiled within chromosomes, the total DNA in a typical eukaryotic cell would extend thousands of times the length of the cell nucleus.

DNA is the genetic material of all cells and contains information necessary for the synthesis of proteins. DNA is composed of two strands of nucleic acids arranged in a double helix. The nucleic acid strands are composed of a sequence of nucleotides. The nucleotides in DNA have four kinds of nitrogen containing bases: adenine (A), guanine (G), cytosine (C), and thymine (T). Within DNA, each strand of nucleic acid is partnered with the other strand by bonds that form between these nucleotides. Complementary base pairing dictates that adenine pairs only with thymine, and guanine pairs only with cytosine (and vice versa). Thus, by knowing the sequence of bases in one strand of the DNA helix, you can determine the sequence on the other strand. For instance, if the sequence in one strand of DNA were ATTCG, the other strand's sequence would be TAAGC.

DNA functions in the cell by providing a template by which another nucleic acid, called ribonucleic acid (RNA), is formed. Like DNA, RNA is also composed of nucleotides. Unlike DNA, RNA is single stranded and does not form a helix. In addition, the RNA bases are the same as in DNA, except that uracil replaces thymine. RNA is transcribed from DNA in the nucleus of the cell. Genes are expressed when the chromosome uncoils with the help of enzymes called helicases and specific DNA binding proteins. DNA is transcribed into RNA.

Newly transcribed RNA is called messenger RNA (mRNA). Messenger RNA leaves the nucleus through the nuclear pore and enters into the cytoplasm. There, the mRNA molecule binds to a ribosome (also composed of RNA) and initiates protein synthesis. Each block of three nucleotides, called codons, in the mRNA sequence encodes for a specific amino acid, the building blocks of a protein.

Genes are part of the DNA sequence called coding DNA. Noncoding DNA represents sequences that do not have genes and only recently have been found to have many new important functions. Out of the 3 billion base pairs that exist in the human DNA, there are only about 40,000 genes. The noncoding sections of DNA within a gene are called introns, while the coding sections of DNA are called exons. After transcription of DNA to RNA, the RNA is processed. Introns from the mRNA are excised out of the newly formed mRNA molecule before it leaves the nucleus.

The human genome (which represents the total amount of DNA in a typical human cell) has approximately 3 × 10⁹ base pairs. If these nucleotide pairs were letters, the genome book would number over a million pages. There are 23 pairs of chromosomes, for a total number of 46 chromosomes in a diploid cell, or a cell having all the genetic material. In a haploid cell, there is only half the genetic material. For example, sex cells (the sperm or the egg) are haploid, while many other cells in the body are diploid. One of the chromosomes in the set of 23 is an X or Y (sex chromosomes), while the rest are assigned numbers 1 through 22. In a diploid cell, males have both an X and a Y chromosome, while females have two X chromosomes. During fertilization, the sex cell of the father combines with the sex cell of the mother to form a new cell, the zygote, which eventually develops into an embryo. If the one of the sex cells has the full complement of chromosomes (diploidy), then the zygote would have an extra set of chromosomes. This is called triploidy and represents an anomaly that usually results in a miscarriage. Sex cells are formed in a special kind of cell division called meiosis. During meiosis, two rounds of cell division ensure that the sex cells receive the haploid number of chromosomes.

Chromosomes can be visible using a microscope just prior to cell division, when the DNA within the nucleus uncoils as it replicates. By visualizing a cell during metaphase, a stage of cell division or mitosis, researchers can take pictures of the duplicated chromosome and match the pairs of chromosomes using the characteristic patterns of bands that appear on the chromosomes when they are stained with a dye called giemsa. The resulting arrangement is called a karyotype. The ends of the chromosome are referred to as telomeres, which are required to maintain stability and recently have been associated with aging. An enzyme called telomerase maintains the length of the telomere. Older cells tend to have shorter telomeres. The telomere has a repeated sequence (TTAGGG) and intact telomeres are important for proper DNA replication processes.

Karyotypes are useful in diagnosing some genetic conditions, because the karyotype can reveal an aberration in chromosome number or large alterations in structure. For example, Down's syndrome is caused by an extra chromosome 21, called trisomy 21. A karyotype of a child with Down's syndrome would reveal this extra chromosome.

A chromosome usually appears to be a long, slender rod of DNA. Pairs of chromosomes are called homologues. Each separate chromosome within the duplicate is called a sister chromatid. The sister chromatids are attached to each other by a structure called the centromere. Chromosomes appear to be in the shape of an X after the material is duplicated. The bottom, longer portion of the X is called the long arm of the chromosome (q-arm), and the top, shorter portion is called the short arm of the chromosome (p-arm).

DNA in chromosomes is associated with proteins and this complex is called chromatin. Euchromatin refers to parts of the chromosome that have coding regions or genes, while heterchromatin refers to regions that are devoid of genes or regions where gene transcription is turned off. DNA binding proteins can attach to specific regions of chromatin. These proteins mediate DNA replication, gene expression, or represent structural proteins important in packaging the chromosomes. Histones are structural proteins of chromatin and are the most abundant protein in the nucleus. In fact, the mass of histones in a chromosome is almost equal to that of DNA. Chromosomes contain five types of these small proteins: H1, H2A, H2B, H3, and H4. There are two of each of latter four histones that form a structure called the octomeric histone core. The H1 histone is larger than the other histones, and performs a structural role separate from the octomeric histone core in organizing DNA within the chromosome.

The octomeric histone core functions as a spool from which DNA is wound two times. Each histone-DNA spool is called a nucleosome. Nucleosomes occur at intervals of every 200 base pairs of the DNA helix. In photographs taken with the help of powerful microscopes , DNA wrapped around nucleosomes resembles beads (the nucleosome) threaded on a string (the DNA molecule). The DNA that exists between nucleosomes is called linker DNA. Chromosomes can contain some very long stretches of linker DNA. Often, these long linker DNA sequences are the regulatory portions of genes. These regulatory portions switch genes on when certain molecules bind to them.

Nucleosomes are the most fundamental organizing structure in the chromosome. They are packaged into structures that are 30 nanometers in size and called the chromatin fiber (compared to the 2 nm DNA double helix, and 11 nm histone core). The 30 nanometer fibers are sometimes then further folded into a larger chromatin fiber that is approximately 300 nanometers thick and represented on of the arms of the chromsome. The chromatin fibers are formed into loops by another structural protein. Each loop contains 20,000–30,000 nucleotide pairs. These loops are then arranged within the chromosomes, held in place by more structural proteins. Metaphase chromosomes are approximately 1400 nm wide.

Chromosomes in eukaryotes perform a useful function during mitosis, the process in which cells replicate their genetic material and then divide into two new cells (also called daughter cells). Because the DNA is packaged within chromosomes, the distribution of the correct amount of genetic material to the daughter cells is maintained during the complex process of cell division.

Before a cell divides, the chromosomes are replicated within the nucleus. In a human cell, the nucleus just prior to cell division contains 46 pairs of chromosomes. When the cell divides, the sister chromatids from each duplicated chromosome separate. Each daughter cell ends up with 23 pairs of chromosomes and after DNA replication, the daughter cells have a diploid number of chromosomes.

In meiosis, the type of cell division that leads to the production of sex cells, the division process is more complicated. Two rounds of cell division occur in meiosis. Before meiosis, the chromosomes replicate, and the nucleus has 46 pairs of chromosomes. In the first round of meiotic cell division, the homologous chromosomes pairs separate as in mitosis (a stage called meiosis I). In the second round of cell division (meisosis II), the sister chromatids of each chromosome separate at the centromere, so that each of the four daughter cells receives the haploid number of chromosomes.

see also DNA; DNA databanks; DNA fingerprint; DNA mixtures, forensic interpretation of mass graves; DNA profiling; Evidence; Gene; STR (short tandem repeat) analysis; War forensics.

REPLICATION

Philosophers have long identified replication as an important facilitator of scientific progress. Several terms have been used to denote the ability to assess past work through replication, including "intersubjective testability," "reliability," and "verifiability by repetition." Authors of scientific papers typically describe the methods and materials they used in their research so that, at least hypothetically, others can repeat the work and reproduce the reported results. Successful replication of their own and others' work gives researchers confidence in its validity and reassures them about the fruitfulness of the general line of inquiry they are following. In contrast, inability to replicate one's own or others' results casts doubt upon the validity of the previous work. Critics argue that because sociologists infrequently attempt to replicate findings, they are both less able to identify valid lines of inquiry and more likely to follow spurious ones.

One can identify a continuum ranging from exact to weakly approximate replication. The former, also called repetition, consists of attempts to use the same materials and procedures as previous research to determine whether the same results can be obtained. Approximate replication, on the other hand, consists of using some but not all of the conditions of a previous study. By systematically varying research conditions in a series of approximate replications, it may be possible to determine the precise nature of a previous study's results and the extent to which they also hold for different populations and situations (Aronson et al. 1998). Researchers usually value successful approximate replication more than successful exact replication because the latter contributes less to existing knowledge.

In the natural sciences, experimentalists are usually expected to carry out successful exact replications of their own work before submitting it for publication. This reduces the likelihood of reporting spurious results and of misleading one's colleagues. Exact replications of others' research are often difficult and costly to execute, however, and natural scientists rarely attempt them except in cases where the original work is theoretically important or has high potential practical value, or where there is suspicion of fraud. Another disincentive for carrying out exact replications of already published work is that such work is usually difficult to publish. This is true not only because little new knowledge results from an exact replication, but also because the meaning of a failure to replicate exactly is often ambiguous. Failures can indicate that the original work was flawed, but they may also be due to inadequate specification of research procedures, the existence of a stochastic element in the production of results, or errors in the replication itself (Harry M. Collins 1985). By contrast, approximate replications, especially those involving the modification of research instruments and their application to new areas of inquiry, are common in the natural sciences, and this has led some to identify them as constituting a central element of "rapid-discovery, high-consensus science" (Randall Collins 1994).

Many hold that social scientists' opportunities to carry out replications, especially exact replications, are severely limited. This is partly because social scientists often use nonexperimental research techniques that are difficult to repeat exactly. In addition, changing social and historical contexts can influence studies' results. As a result, failures to obtain the same results as reported by previous studies are even more ambiguous in the social sciences than in the natural sciences (Schuman and Presser 1981). This ambiguity may account for social scientists' continued interest in concepts and theories stemming from studies whose results have repeatedly failed to be replicated (e.g., sex differences in fear of success and patterns of moral development).

Nevertheless, critics have long argued that behavioral scientists need to attempt more replications of previous research because their dependence on statistical inference produces many spurious reports of "statistically significant" results. Statistical inference allows researchers only to reject or fail to reject a null hypothesis. Each of these two outcomes is subject to error due to the probabilistic nature of statistical hypothesis testing; sometimes researchers reject null hypotheses that are actually true (type one error), and sometimes they fail to reject null hypotheses that are actually false (type two error). However, failure to reject a null hypothesis does not justify accepting it, and studies that do not yield rejections therefore are often judged as contributing little. As a result, scholarly journals tend to publish only papers that report the rejection of null hypotheses, some of which are the result of type one errors (Sterling 1959). Furthermore, to ensure that they will be able to reject null hypotheses, researchers sometimes use inappropriate analytic procedures that maximize their chances of obtaining statistically significant results (Selvin and Stuart 1966), increasing the likelihood that published findings are due to type one errors. To counteract these patterns, some have argued that behavioral science editors should set aside space in their journals for the publication of replication attempts, and to publish studies that fail to replicate earlier results even when the replications themselves fail to reject null hypotheses.

Despite the calls for increased replication, behavioral science journals publish few papers reporting replication attempts. In an early examination of this issue, Sterling (1959) reported that among 362 articles in psychology journals, 97 percent of those reporting a test of significance rejected the null hypothesis, but that none was an explicit replication. Ironically, many have replicated Sterling's results (cf. Dickersin 1990; Gaston 1979; Reid et al. 1981). These studies probably underestimate the prevalence of replication, because they do not count papers reporting a set of experiments that comprise both an original result and one or more approximate replications of it. By not encouraging more replication, however, behavioral science journals may foster elaborate and vacuous theorizing at the expense of identifying factual puzzles that deserve theoretical analysis (Cook and Campbell 1979, p. 25).

Although the traditional view of replication entails the collection of new data—including data on additional cases or additional measures—statisticians and social scientists have suggested alternative replication strategies. One is to build replication into a study from the start. For example, a researcher can draw a sample large enough to allow its random partition into two subsamples. Data from one subsample can then be used to check conclusions drawn on the basis of analyses of data from the other. Another approach, requiring the intensive use of computing resources, is to draw multiple random subsamples from already collected data and then use these subsamples to crossvalidate results (Finifter 1972). This general strategy, which includes such techniques as "jackknifing" and "bootstrapping," is also used to assess sampling variances for complex sampling designs (see Sampling Procedures). Still another elaboration of the basic idea of replication is the general approach called meta-analysis. Here the analyst treats previous studies on a topic or relationship as a sample of approximate replications. By statistically analyzing whether and how studies' results vary, one can determine how generalizable a finding is and the extent to which differences in study design account for variation in results (Hunter and Schmidt 1990). Finally, replication may also be fostered by the increased availability of already-collected data sets stemming from the establishment of data depositories, and funding agency requirements that data from supported projects be made accessible to other researchers. Access to previously collected data makes it possible to carry out both exact replications of previous analyses and approximate replications that alter the analytic procedures used by the original researcher.

(see also: Sampling Procedures)

references

Aronson, Elliot, Timothy D. Wilson, and Marilynn B. Brewer 1998 "Experimentation in Social Psychology." In Daniel T. Gilbert, Susan T. Fiske, and Gardner Lindzey, eds., Handbook of Social Psychology, 4th ed. Boston: McGraw-Hill.

Collins, Harry M. 1985 Changing Order: Replication andInduction in Scientific Practice. London: Sage.

Collins, Randall 1994 "Why the Social Sciences Won't Become High-Consensus, Rapid-Discovery Science." Sociological Forum 9:155–177.

Cook, Thomas D., and Donald T. Campbell 1979 Quasi-Experimentation: Design and Analysis Issues for FieldStudies. Chicago: Rand-McNally.

Dickersin, Kay 1990 "The Existence of Publication Bias and Risk Factors for Its Occurence." Journal of theAmerican Medical Association 263:1385–1389.

Finifter, Bernard M. 1972 "The Generation of Confidence: Evaluating Research Findings by Random Subsample Replication." In Herbert L. Costner, ed., Sociological Methodology 1972. San Francisco: Jossey-Bass.

Gaston, Jerry 1979 "The Big Three and the Status of Sociology." Contemporary Sociology 8:789–793.

Hunter, John E., and Frank L. Schmidt 1990 Methods ofMeta-Analysis. Newbury Park, Calif.: Sage.

Reid, L. H., L. C. Soley, and R. D. Rimmer 1981 "Replications in Advertising Research: 1977, 1978, 1979." Journal of Advertising 10:3–13.

Schuman, Howard, and Stanley Presser 1981 "Mysteries of Replication and Non-Replication." In Howard Schuman and Stanley Presser, eds. Questions andAnswers in Attitude Surveys. New York: Academic Press.

Selvin, Hannan C., and Alan Stuart 1966 "Data-Dredging Procedures in Survey Analysis." American Statistician 20:20–23.

Sterling, Theodore D. 1959 "Publication Decisions and Their Possible Effects on Inferences Drawn from Tests of Significance—Or Visa Versa." Journal of theAmerican Statistical Association 54:30–34.

Lowell L. Hargens

replication Repeating a study, in exactly the same format, to check whether the same results are obtained a second or subsequent time (usually by a different researcher). One variation is simultaneous replication of the same study, either in the same context, or in contexts carefully selected for their known variation on one key variable. There can also be replication via secondary analysis of a major data-set, to re-test hypotheses, and to assess the impact of the particular program or software employed in the original analysis.

Arguably, and for a variety of reasons, not nearly enough resources in the discipline are devoted to replicating results: the effort, cost, and time involved in conducting social surveys usually preclude systematic replication; funding agencies are less likely to pay for investigations which seek only to confirm earlier findings; and there is little kudos for researchers (and, one suspects, not much career advancement) to be had in reproducing existing studies. (In psychology, by comparison, the dominant experimental methods lend themselves to the widespread use of replication studies.) However, although whole surveys are all too infrequently replicated, individual questions and batteries of questions often are—especially where these have been assembled into scales (for example to measure attitudes or personality traits).

Strict replication shades gradually into the idea of a ‘re-study’. Re-studies are also in a sense replications, but are usually conducted at time intervals such that observable differences from earlier findings can reasonably be attributed to real change in the subjects or processes under investigation, rather than to either chance or measurement error in the original study. Prominent and informative re-studies include the two community studies of the English town of Banbury, conducted by Margaret Stacey and her colleagues in 1948–51 and 1966–8 (compare Tradition and Change, 1960, and Power, Persistence and Change, 1975), and the studies by Robert Redfield and Oscar Lewis of Tepoztlan in Mexico (in 1926 and 1943 respectively). Redfield depicts the community in question as an ideal-typical type of folk society (smooth functioning, well-integrated, contented, well-adjusted) whereas Lewis paints a picture dominated by individualism, fear, lack of co-operation, envy, schism, and mistrust. Each party accused the other of methodological failings (for example asking the wrong questions of the wrong informants). The celebrated disjuncture between the two accounts was eventually resolved, when a further investigation revealed that the society had in all probability changed dramatically in the intervening years, due to increases in population pressure and a programme of radical land redistribution (see P. Coy , ‘A Watershed in Mexican Rural History’, Journal of Latin American Studies, 1971
). See also RELIABILITY.