Kilobase Pairs In Size Research Articles

Long-Read DNA Sequencing: Recent Advances and Remaining Challenges.

DNA sequencing has revolutionized medicine over recent decades. However, analysis of large structural variation and repetitive DNA, a hallmark of human genomes, has been limited by short-read technology, with read lengths of 100-300 bp. Long-read sequencing (LRS) permits routine sequencing of human DNA fragments tens to hundreds of kilobase pairs in size, using both real-time sequencing by synthesis and nanopore-based direct electronic sequencing. LRS permits analysis of large structural variation and haplotypic phasing in human genomes and has enabled the discovery and characterization of rare pathogenic structural variants and repeat expansions. It has also recently enabled the assembly of a complete, gapless human genome that includes previously intractable regions, such as highly repetitive centromeres and homologous acrocentric short arms. With the addition of protocols for targeted enrichment, direct epigenetic DNA modification detection, and long-range chromatin profiling, LRS promises to launch a new era of understanding of genetic diversity and pathogenic mutations in human populations.

DNA in nanochannels: theory and applications.

Nanofluidic structures have over the last two decades emerged as a powerful platform for detailed analysis of DNA on the kilobase pair length scale. When DNA is confined to a nanochannel, the combination of excluded volume and DNA stiffness leads to the DNA being stretched to near its full contour length. Importantly, this stretching takes place at equilibrium, without any chemical modifications to the DNA. As a result, any DNA can be analyzed, such as DNA extracted from cells or circular DNA, and it is straight-forward to study reactions on the ends of linear DNA. In this comprehensive review, we first give a thorough description of the current understanding of the polymer physics of DNA and how that leads to stretching in nanochannels. We then describe how the versatility of nanofabrication can be used to design devices specifically tailored for the problem at hand, either by controlling the degree of confinement or enabling facile exchange of reagents to measure DNA-protein reaction kinetics. The remainder of the review focuses on two important applications of confining DNA in nanochannels. The first is optical DNA mapping, which provides the genomic sequence of intact DNA molecules in excess of 100 kilobase pairs in size, with kilobase pair resolution, through labeling strategies that are suitable for fluorescence microscopy. In this section, we highlight solutions to the technical aspects of genomic mapping, including the use of enzyme-based labeling and affinity-based labeling to produce the genomic maps, rather than recent applications in human genetics. The second is DNA-protein interactions, and several recent examples of such studies on DNA compaction, filamentous protein complexes, and reactions with DNA ends are presented. Taken together, these two applications demonstrate the power of DNA confinement and nanofluidics in genomics, molecular biology, and biophysics.

Engineering Synthetic Chromosomes by Sequential Loading of Multiple Genomic Payloads over 100 Kilobase Pairs in Size

Gene delivery vehicles currently in the clinic for treatment of monogenic disorders lack sufficient carrying capacity to efficiently address complex polygenic diseases. Thus, to engineer multifaceted genetic circuits for bioengineering human cells as a therapeutic option for polygenic diseases, we require new tools that are currently in their infancy. Mammalian artificial chromosomes, or synthetic chromosomes, represent a viable approach for delivery of large genetic payloads that are mitotically stable and remain independent of the host genome. Previously, we described a mammalian synthetic chromosome platform, termed the ACE system, that requires a single unidirectional integrase for the introduction of multiple genes onto the ACE platform chromosome. In this report, we provide a proof of concept that the ACE synthetic chromosome bioengineering platform is amenable to sequential delivery of off-the-shelf large genomic fragments. Specifically, large genomic clones spanning the human solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1 or GLUT1, 169 kbp), and human monocarboxylate transporter 1 (SLC16A1 or MCT1, 144 kbp) genetic loci were engineered onto the ACE platform and demonstrated to express and correctly splice both gene transcripts. Thus, the ACE system provides a facile and tractable engineering platform for the development of gene-based therapeutic agents targeting polygenic diseases.

Microfluidic long DNA sample preparation from cells.

A number of outstanding problems in genomics, such as identifying structural variations and sequencing through centromeres and telomeres, stand poised to benefit tremendously from emerging long-read genomics technologies such as nanopore sequencing and genome mapping in nanochannels. However, optimal application of these new genomics technologies requires facile methods for extracting long DNA from cells. These sample preparation tools should be amenable to automation and minimize fragmentation of the long DNA molecules by shear. We present one such approach in a poly(dimethylsiloxane) device, where gel-based high molecular weight DNA extraction and continuous flow purification in a 3D cell culture-inspired geometry is followed by electrophoretic extraction of the long DNA from the miniaturized gel. Molecular combing reveals that the device produces molecules that are typically in excess of 100 kilobase pairs in size, with the longest molecule extending up to 4 megabase pairs. The microfluidic format reduces the standard day-long and labor-intensive DNA extraction process to 4 hours, making it a promising prototype platform for routine long DNA sample preparation.

Cardiovascular RNA Interference Therapy

Understanding of the roles of noncoding RNAs (ncRNAs) within complex organisms has fundamentally changed. It is increasingly possible to use ncRNAs as diagnostic and therapeutic tools in medicine. Regarding disease pathogenesis, it has become evident that confinement to the analysis of protein-coding regions of the human genome is insufficient because ncRNA variants have been associated with important human diseases. Thus, inclusion of noncoding genomic elements in pathogenetic studies and their consideration as therapeutic targets is warranted. We consider aspects of the evolutionary and discovery history of ncRNAs, as far as they are relevant for the identification and selection of ncRNAs with likely therapeutic potential. Novel therapeutic strategies are based on ncRNAs, and we discuss here RNA interference as a highly versatile tool for gene silencing. RNA interference-mediating RNAs are small, but only parts of a far larger spectrum encompassing ncRNAs up to many kilobasepairs in size. We discuss therapeutic options in cardiovascular medicine offered by ncRNAs and key issues to be solved before clinical translation. Convergence of multiple technical advances is highlighted as a prerequisite for the translational progress achieved in recent years. Regarding safety, we review properties of RNA therapeutics, which may immunologically distinguish them from their endogenous counterparts, all of which underwent sophisticated evolutionary adaptation to specific biological contexts. Although our understanding of the noncoding human genome is only fragmentary to date, it is already feasible to develop RNA interference against a rapidly broadening spectrum of therapeutic targets and to translate this to the clinical setting under certain restrictions.

Novel precursor-derived Si–C–N ceramic material for purification application

Metagenomic DNA (gDNA) is an important research topic because uncultivated microorganisms represent an interesting reservoir of genes with potential biotechnological applications. Here we describe a novel Si–C–N chromatography approach of gDNA extraction from environmental samples. Amorphous Si–C–N ceramic was obtained by the pyrolysis of a polymer precursor (polycarbosilazane) at 1350 °C in Ar. The purified gDNA fragments were at least 12 kilo base pairs in size and were sufficiently free of contaminants, thus were applicable to both restriction enzyme digestion using five different enzymes and polymerase chain reaction amplification for detecting phylogenetic groups of native microorganisms in environmental samples. This Si–C–N material produces pure gDNA that can be utilized in some of the most common molecular biological procedures applied in the purification step.

Transcriptional Regulation of Rat Scavenger Receptor Class B Type I Gene

The scavenger receptor class B type I (SR-BI) mediates the selective transport of lipids from high density lipoprotein to cells and plays an important role in the reverse uptake of cholesterol to the liver and in the delivery of substrates for steroidogenesis in steroidogenic organs. We report here on the isolation and characterization of the upstream promoter region of the rat SR-BI gene. The transcription start site for rat SR-BI was mapped, and DNA sequence analysis revealed the presence of binding sites for the Sp1 family in the proximal 5'-flanking region. Analysis of deletion mutants with different 5' lengths revealed that the region between -121 and -90 base pairs from the transcription start site is essential for the efficient transcription of SR-BI. Both Sp1 and Sp3 bind to three GC boxes in the region (-141 to -1 base pairs) in a sequence-specific manner. Mutations in any of the GC boxes decreased efficient transcription from this promoter in MA-10 mouse Leydig tumor cells. The overexpression of Sp1 or Sp3 protein enhanced the rat SR-BI promoter activity. These results indicate that Sp1 family members of transcription factors are essential for transcription of the rat SR-BI gene.

Multiple replicons constitute the 6.5-megabase genome of Burkholderia pseudomallei

Burkholderia pseudomallei is a causative agent of melioidosis, a fatal tropical infectious disease endemic in Southeast Asia and Northern Australia. In order to determine the size and characteristics of the bacterial genome, the B. pseudomallei genome and genes were analyzed by pulsed field gel electrophoresis of the undigested, intact megabase DNA, and by computational analysis of nucleotide sequences of B. pseudomallei genes which have been sequenced by several investigators and already deposited in a public database. The results showed that the B. pseudomallei genome consists of two large replicons, and that both contain ribosomal RNA gene sequences, indicating the presence of two chromosomes. The classical arabinose-negative B. pseudomallei isolate K96243 has chromosomes of approximately 3563±73 and 2974±40 kilobase-pairs in size, giving a total genome size of about 6.5 million base-pairs. The arabinose-positive nonvirulent biotype of B. pseudomallei also has two replicons which are smaller than those of the arabinose-negative biotype. Analysis of the publicly-available nucleotide sequences showed that the average B. pseudomallei gene is approximately 1031 base-pairs in size, with an average G+C content of 65.7%. The genome is gene-rich and about 89% of the coding capacity is used as coding sequences. It can therefore be estimated that the entire B. pseudomallei genome encodes about 5600 genes.

Double-stranded RNA and associated virulence in South African isolates of Sphaeropsis sapinea

Sphaeropsis sapinea (Fr.) Dyko & Sutton is an opportunistic pathogen of various Pinus spp., causing severe shoot blight and dieback. Some isolates of S. sapinea display characteristics such as reduced virulence, reduced growth rate, lack of pigmentation, altered colony morphology, and suppressed conidiation. South African isolates of S. sapinea displaying a range of growth patterns, including reduced growth rate and atypical morphology, were screened for the presence of double-stranded RNA (dsRNA). They were also tested for relative virulence in pathogenicity tests. Double-stranded RNA was isolated by means of phenol extraction and cellulose chromatographic purification. A single species of dsRNA (± 4.3 kilobase pairs in size) was obtained from two slow-growing isolates and two isolates with more regular growth. The virulence of these dsRNA-containing isolates was tested on mature Pinus patula Schlecht. et Cham. trees. Although reduced virulence was positively correlated with slower growth in vitro, the presence of dsRNA could not be linked to either of these characteristics.Key words: double-stranded RNA, hypovirulence, Sphaeropsis sapinea.

Identification and Characterization of a New Latent Transforming Growth Factor-β-binding Protein, LTBP-4

Transforming growth factor betas (TGF-betas) are secreted by most cell types as latent high molecular weight complexes consisting of TGF-beta and its latency associated peptide (LAP) propeptide dimers, covalently linked to latent TGF-beta-binding proteins (LTBPs). Currently, three different LTBPs are known (LTBPs 1, 2, and 3), all with highly similar protein domain structure consisting of epidermal growth factor-like and 8-Cys repeats. The 3rd 8-Cys repeat of LTBP-1 mediates its association with TGF-beta1.LAP. By using an expressed sequence tag homologous to the 3rd 8-Cys repeat of human LTBP-1 as a probe, a novel cDNA similar to known LTBPs was cloned from human heart cDNA library. This cDNA was named LTBP-4 and found to exist in at least four different forms, generated by alternative splicing at the amino terminus and at the central epidermal growth factor repeat domain. One of the alternative amino-terminal forms contained an RGD sequence, indicating possible cell-surface interactions with integrins. LTBP-4 gene was localized to chromosomal position 19q13. 1-19q13.2. The major LTBP-4 mRNA form is about 5.1 kilobase pairs in size and is predominantly expressed in the heart, aorta, uterus, and small intestine. Immunoblotting analysis indicated that LTBP-4 was secreted from cultured human lung fibroblasts both in a free form and in a disulfide bound complex with a TGF-beta. LAP-like protein. Both LTBP-4 forms were also found to be deposited in the extracellular matrix. The matrix-associated LTBP-4 was susceptible to proteolytic release with plasmin. LTBP-4 is a new member of the growing LTBP-fibrillin family of proteins and offers an alternative means for the secretion and targeted matrix deposition of TGF-betas or related proteins.

Cloning and Chromosomal Location of a Novel Member of the Myotonic Dystrophy Family of Protein Kinases

We have cloned a novel serine/threonine protein kinase (PK428) which is highly related (65%) within the kinase domain to the myotonic dystrophy protein kinase (DM-PK), as well as the cyclic AMP-dependent protein kinase (33%). Northern blots demonstrate that PK428 mRNA is distributed widely among tissues and is expressed at the highest levels in pancreas, heart, and skeletal muscle, with lower levels in liver and lung. Two PK428 mRNAs 10 and 3.8 kilobase pairs in size are seen in a number of cell lines, including hematopoietic and breast cancer cells. An antibody generated to a glutathione S-transferase-PK428 fusion protein detects a 65-kDa protein in these cell lines, and a similarly sized protein when the cloned cDNA is transiently expressed in Cos 7 cells. Immunoprecipitation of the transiently expressed PK428 protein and incubation with [gamma-32P]ATP demonstrate that it is capable of autophosphorylation. In addition, immunoprecipitates of the PK428 protein kinase also phosphorylated histone H1 and a peptide encoding a cyclic AMP-dependent protein kinase substrate. The gene corresponding to the 3.8-kb PK428 mRNA, and its corresponding 65-kDa protein, was isolated by polymerase chain reaction screening of a P1 phage human genomic library. Using this P1 phage clone as a probe, the PK428 gene was located on 1q41-42, a possible location for a human senescence gene, a gene associated with Rippling muscle disease, as well as a region associated with genetically acquired mental retardation.

Genome Structure of a Virus Infecting the Marine Brown Alga Ectocarpus siliculosus

We describe a procedure for the isolation of virus particles from the marine brown alga Ectocarpus siliculosus. Virus particles are composed of at least 13 different polypeptides, including two glycoproteins, and double-stranded DNA. A typical virus DNA preparation contains three fractions, namely linear DNA and circular DNA, each composed of about 320 kilobase pairs, as well as DNA fragments, 10 to 60 kilobase pairs in size. The large linear and the circular DNA contain single-stranded regions (average length: 2.9 kilobases). We propose that the native Ectocarpus virus genome is a circular DNA molecule whose double strand is interrupted by single-stranded regions. During the preparation procedure, the DNA circles tend to break at the single-stranded sites producing large linear as well as fragmented DNA.

Microinjection of Intact 200- to 500-kb Fragments of YAC DNA into Mammalian Cells

DNA of yeast artificial chromosomes (YACs) was prepared for microinjection by separation from most of the natural yeast chromosomes on a pulsed-field gel, treatment with agarase, and centrifugation. A salt concentration of 100 mM NaCl was necessary to protect the DNA from shear during these procedures. Injection of a 590-kb YAC, yGART2, into Chinese hamster ovary cells gave rise to cells expressing the 40-kb human GART gene carried on the YAC. Nine of 12 cell lines analyzed contained an intact stretch of at least 110 kb of YAC DNA surrounding the GART gene, and one cell line contained at least 480 kb, but not the entire 590 kb, intact. Mouse L A-9 cells were similarly injected with DNA of a 230-kb YAC containing the human beta-globin gene cluster and a mammalian selectable marker. Seven of 10 of the resulting cell lines contained both YAC vector arms plus the intact 140-kb SfiI fragment spanning the beta-globin gene. Three cell lines were analyzed by RecA-assisted restriction endonuclease (RARE) cleavage and found to contain the entire intact 210-kb YAC insert. Introduction of similarly prepared DNA into mammalian cells by lipofection gave rise to cell lines with multiple YAC fragments that were generally shorter than the YAC fragments found in microinjected cell lines. The results show that microinjection of gel-purified YAC DNA into mammalian cells is an efficient method of transferring DNA fragments several hundred kilobase pairs in size into mammalian cells.

Nucleotide Sequence of the psbB Gene of Chlamydomonas reinhardtii Chloroplasts

The chloroplast psbB gene encodes the 47 kD Chl-binding protein associated with the PSII reaction center (Table I). The nucleotide sequence of psbB has been determined for several vascular plants (2, 6, 7), Euglena gracilis (3), Synechocystis (9), and Anabaena (5). The psbB gene of Euglena contains five introns, whereas the gene from the other eukaryotic organisms lack introns. The psbB locus exhibits a complex pattern of transcription and processing in spinach (10), maize (1), and Marchantia (4). The spinach psbB gene is co-transcribed with psbH, petB, and petD; the latter two genes contain introns. The psbB gene of the liverwort Marchantia is cotranscribed with ORF43, psbH, petB, and petD. A gene encoding 35 amino acids (ORF35) is localized between psbB and ORF43, but it is transcribed from the opposite strand. Although the organization of the chloroplast genome is highly conserved among most plants, the organization of the Euglena and Chiamydomonas chloroplast is somewhat different. In fact, petB and petD are not co-transcribed with psbB in Chlamydomonas: Sieburth et al. (8) have demonstrated that petB and petD are transcribed and translated normally while psbB mRNA, and several other transcripts encoded by adjacent loci, are rapidly degraded in Chlamydomonas nuclear mutant GE2. 10. During the analysis of this psbB mutant, we became interested in more precise mapping and sequencing of the wild-type Chlamydomonas psbB gene to determine if there are unique structural features of the transcript. The psbB gene is localized primarily on clone P62, an EcoR 1 fragment of approximately 10 kilobase pairs, from the Chlamydomonas chloroplast (8). An EcoR 1 -Pst 1 fragment was subcloned to generate clone 4-8 for further analysis. Additionally, the neighboring EcoRl clone pEC51 also encodes a portion of the psbB gene (8). A Pst 1 -EcoR 1 chloroplast fragment contained within this larger EcoR 1 fragment was also subcloned. This clone, denoted cp211 9A, is approximately 1.2 kilobase pairs in size and contains the region that encodes the very 3'-most portion of psbB. The coding sequence of this gene is 1524 base pairs for a polypeptide of 508 amino acids (Fig. 1). There are no introns in this algal gene. The predicted amino acid sequence is 80% similar to that of spinach and 76% similar to that of Euglena. There are three segments of the untranslated regions of the locus that Table I. Characteristics of psbB from Chlamydomonas reinhardtii

Characterization of Satellite DNA from Three Marine Dinoflagellates (Dinophyceae): Glenodinium sp. and Two Members of the Toxic Genus, Protogonyaulax

Using CsCl-Hoechst dye or CsCl-ethidium bromide gradients, satellite and nuclear DNAs were separated and characterized in three marine dinoflagellates: Glenodinium sp., and two toxic dinoflagellates, Protogonyaulax tamarensis and Protogonyaulax catenella. In all three dinoflagellates, the lowest density fraction, satellite DNA(1), hybridized to chloroplast genes derived from terrestrial plants and/or other algae. Dinoflagellate chloroplast DNAs exhibited molecular sizes of 114 to 125 kilobase pairs, which is consistent with plastid sizes determined for other chromophytic algae (120-150 kilobase pairs). Mitochondrial DNA was not resolved from nuclear DNA in this system. Two additional satellite DNAs, satellite DNA(2) and satellite DNA(3), recovered from P. tamarensis and P. catenella were similar to one another, both within and between species, when characterized by restriction enzyme analysis. These satellites were 85 to 95 kilobase pairs in size, and exhibited restriction fragments that hybridized to yeast nuclear ribosomal RNA genes. Restriction enzyme analyses and DNA hybridization studies of cpDNA document that the two Protogonyaulax isolates are not evolutionarily identical.

Molecular cloning of the murine ST2 gene. Characterization and chromosomal mapping

The genomic locus of the murine ST2 gene was isolated based on homology with a murine ST2 complementary DNA sequence and its complete nucleotide sequence was determined. The locus is composed of eight exons and seven introns and is approx. 9 kilobase pairs in size. Two Sp1 binding sites are present in the 5' flanking region. The murine ST2 gene, which was expressed only in the growth-stimulated BALB/c-3T3 cells, was mapped to mouse chromosome one, very tightly linked to the interleukin 1 receptor-type 1 locus.

Characterization of cDNA and genomic sequences encoding a chicken phospholamban.

We report on the isolation and characterization of cDNA and genomic sequences encoding a chicken cardiac phospholamban. Three mRNAs, 0.6, 1.1, and 3.3 kilobase pairs in size, were detected in cardiac and slow-tonic muscle RNAs on Northern blots. We determined that these mRNAs differ in the length of their 3'-untranslated regions, perhaps because they utilize alternative polyadenylation signals. We studied the developmental and tissue-specific expression of phospholamban mRNA using nuclease mapping analysis. There appeared to be considerable homology between coding and 3'-untranslated regions of phospholamban mRNAs from cardiac and slow-tonic muscles during development, strongly suggesting that these mRNAs are encoded by the same gene. The phospholamban gene, which is present as a single copy in the chicken genome, is about 10 kilobase pairs long, with approximately 6.5 kilobase pairs representing the intron sequences. Primer extension and nuclease mapping analyses of the 5' end of the phospholamban mRNA showed that the three transcripts are initiated from the same initiation site. Analysis of the putative promoter region revealed "TATA box" and "CAAT box" sequences, putative muscle-specific elements, and a cyclic AMP-responsive element.

Comparison of Haemophilus paragallinarum isolates by restriction endonuclease analysis of chromosomal DNA

Chromosomal DNA from Haemophilus paragallinarum was examined by restriction endonuclease analysis (REA) using the enzymes BamHI, EcoRI, HindIII, or SmaI. The enzyme SmaI had no apparent effect upon the DNA from eight representative H. paragallinarum isolates. The remaining enzymes vut the H. paragallinarum DNA to varying degrees, with the most useful patterns for distinguishing isolates being given by HindIII. The REA patterns given by HindIII were stable under both in vitro and in vivo conditions. The use of the enzyme HindIII showed that eight Australian isolates of H. paragallinarum were genetically similar. In contrast, 14 isolates of H. paragallinarum from outside Australia were markedly different from each other and the Australian isolates. A plasmid of approximately 6 kilobase pairs in size was found in one isolate of H. paragallinarum.

Characterization of the human platelet glycoprotein IIIa gene. Comparison with the fibronectin receptor beta-subunit gene.

This study was designed to determine the structure of the gene for glycoprotein (GP) GPIIIa, the beta-subunit of the platelet membrane GPIIb-IIIa complex. The complexity of the gene was determined after Southern analysis of human chromosomal DNA. Overlapping genomic clones were isolated from cosmid and phage lambda libraries that contained the entire coding unit of the human gene for the mature GPIIIa protein. The genomic clones spanned approximately 60 kilobase pairs of human DNA sequence. The exon containing segments of the clones was mapped and the exons, including the exonintron junctions, were sequenced. The GPIIIa protein is divided into 14 exons ranging in size from 87 to 430 nucleotides separated by introns, which were 0.3 to 9 kilobase pairs in size. The 3' exon was larger than 1700 nucleotides and contained the 3'-untranslated region. Several structural domains of the GPIIIa protein were contained within individual exons. These included (i) the transmembrane spanning segment, (ii) the cytoplasmic region containing the potential phosphorylation sites, and (iii) the six domains in the NH2-terminal half of GPIIIa that are highly conserved between two other integrin beta-subunits. In contrast, other domains such as the four cysteine-rich repeats were interrupted by introns. Genomic clones for the beta-subunit of the fibronectin receptor (beta 1) were also isolated, partially mapped, and sequenced. Of the eight splice sites identified in beta 1, six occurred at the same amino acid residue in GPIIIa. These results provide genetic evidence that GPIIIa and beta 1 have a common evolutionary origin within the integrin family.

Cloning, Sequence Analysis, and Expression of a cDNA Encoding a Plastid-Localized Heat Shock Protein in Maize

We have cloned and characterized a cDNA encoding a maize (Zea mays L.) heat shock protein (HSP), HSP26. The mRNA of HSP26 is present as a single mRNA species of 1.1 kilobase pairs in size and is detectable when maize seedlings are treated at 40 degrees C but not at 28 degrees C. Accumulation of HSP26 mRNA was detected after 10 minutes of incubation at 40 degrees C, reaching the maximum level after 1 hour. Comparison of the deduced amino acid sequence of maize HSP26 to other HSPs indicated a strong homology to the sequences of two nuclear encoded HSPs that are transported into the chloroplasts during heat shock: pea HSP21 and soybean HSP22. Maize HSP26 was also found to cross-react with anti-pea chloroplast HSP21 antibodies. Because of the sequence homology between maize HSP26, soybean HSP22, and pea HSP21, in vitro chloroplast protein import experiments were conducted. The in vitro synthesized maize HSP26 is specifically imported to the soluble fraction of the chloroplast and processed to a smaller polypeptide. The sequence homology and antibody cross-reactivity between maize HSP26 and pea HSP21 have allowed us to conclude that maize HSP26 is a nuclear-encoded, plastid-localized protein in maize.