Tom Strachan • Andrew Read

Human Molecular GeneticsFourth Edition

Copyright © Garland Science 2011

Chapter 9Organization of the Human Genome

• Mitochondrial genome: 16,569 bp, 37 genes, 44% (G+C), Heavy strand (rich in G), Light strand (rich in C) , and a small section of the genome (7S DNA) is triple stranded (due to repetitive synthesis). 7S contains many of the control sequences and so is called CR/D-loop region.

• Human cells vary in the number of mt DNA molecules (typically thousands of copies/cell).

• Sperms do not contribute mtDNA to the zygote (strictly maternal). During mitosis, mitochondria are passed on to daughter cells by random assortment.

• Mt DNA contains 37 genes, 28 use H strand (rich in G) as their sense strand and 9 use L strand (rich in C).

• Of the 37 mt genes: 22 are tRNA genes; 2 rRNA (23S rRNA and 12S rRNA); 13 are polypeptide coding (oxidative phosphorylation).

• Because mt DNA encodes 13 proteins only, its genetic code has drifted from the universal genetic code.

• 93% of mt DNA is coding, all genes lack introns, for some coding sequences are overlapping, some lack stop codons (added post-transcriptionally), replication of H strand starts at the D loop unidirectionally and 2/3 into the mtDNA replication shifts to using the L strand from a new origin of replication and it proceeds in the opposite direction.

• Nuclear genome: - 3100 Mb (3.1 Gb), more than 26,000 genes (6 of which are RNA genes), ~5% highly conserved including 1.1% protein coding DNA and 4% of conserved untranslated & regulatory sequences. - The coding sequence is present in families of related sequences generated by gene duplication which resulted in pseudogenes and gene fragments. - The 95% non-coding DNA of the human genome is made up of tandem repeats (head to tail) or dispered repeats resulting from retrotransposition of RNA transcripts.

for euchromatic component • Human genome consists of 24 different DNA molecules making 24

chromosomes.

- content: DNA, RNA, histones, non-histones.

- divided to the gene-rich transcriptionally-active euchromatic regions (2.9 Gb) which was used in the Human Genome Project and constitutive heterochromatin (200Mb) which is transcriptionally- inactive composed of long arrays of highly repetitive DNA which are difficult to sequence so are the long arrays of tandemly repeated transcription units encoding 28S, 18S, & 5.8S (were not sequenced as well). Each chromosome has some constitutive heterochromatin at the centromere but chromosomes 1, 9, 16 & 19 have significant heterochromatin in the euchromatic region close to the centromere. Also significant heterochromatin is found in Y and the acrocentric chromosomes 13, 14, 15, 21, & 22.

- Base composition:

Average GC = 41% for euchromatic componenet but there is considerable variation between chromosomes (38% G+C) for chrm. 4 & 13 and 49% for 19.

Giemsa bands (dark bands, low GC, 37%; light bands, hi GC 45%).

CpG dinucleotides, why are they depleted from vertebrate DNA?

• Human gene number:

- At least 26,000 (6000 of which are RNA genes

- C. elegans (1 mm long worm) has 959 somatic cells, genome is 1/30 that of humans, contains 19,099 protein-coding genes & >1000 RNA-coding genes.

Therefore, genome complexity is not parallel to biological complexity.

• Human gene distribution:

- CpG islands are known to strongly associated with genes. Done by hybridizing CpG islands to metaphase chromosomes. The results showed that gene density is high in subtelomeric regions & that some chromosomes (19 & 22) are gene rich while others are gene poor (X & 18).

- Gene density correlates with Giemsa banding. Dark bands are low in G+C content and vise-versa is true for light bands.

• Duplication of DNA segments resulted in copy-number variation and gene families

- Tandem gene duplication: Arise by unequal crossover between unequally aligned chromatids either on homologous chromosomes (unequal crossver) or on the same chromosome (unequal sister chromatid exchange (Fig. 9.5).

- Duplicative transposition: this involves retrotransposition

- Gene duplication by ancestral cell fusion: Invasion by a prokaryotic cell to a eukaryotic cell resulted in establishing organelles. By time pieces of organelle genomes have been excised and transferred to the nuclear genome. This resulted in duplication of cytoplasm encoding genes in the nuclear genome.

- Large-scale subgenomic duplications: arise by chromosome translocations (segmental duplication) (Fig 9.6).

- Whole genome duplication: Comparative genomics studies confirmed that during eukaryote evolution (e.g. chordates).

• Organization, distribution & function human protein-coding genes:

- Human genes show enormous variation in size and internal organization. E.g. Dystrophin gene 2.4 Mb is transcribed in 16 hours

- Diversity in exon-intron organization: very small number of genes lack introns. For intron-containing genes, there is an inverse correlation between gene size and fraction of coding DNA (Table 9.4).This is not because exons in large genes are smaller than those in small genes but because large genes

have huge introns.

- Diversity in repetitive DNA content: gene have repetitive DNA within introns, flanking sequences, and to different extents in

coding sequences.

- Different proteins can be specified by overlapping transcription units:

- Overlapping genes and genes-within-genes: Gene density varies between chromosomes and within regions of same

chromosome.

- In regions with high gene density, overlapping genes maybe found which are typically transcribed from opposing DNA strands

e.g. HLA complex (Fig 9.7A)

- 9% of the humna protein-coding genes overlap and more of 90% of such overlaps involve transcription from opposing strands. However, sometimes small protein-encoding genes are

located within the introns of larger genes e.g. neurofibromatosis type I (NF1) (Fig. 9.7B)

- Some protein-coding genes share a common promoter and are transcribed in opposite directions.

- Protein-coding genes often belong to families that are clustered or

dispersed on multiple chromosomes:

- Examples of clustered gene families in Fig 9.8 while some gene families have copies at two or more chromosomal locations

without gene clustering (Table 9.6).

- Three different classes of gene family according to the extent of sequence identity and structural similarity of the protein products:

1- High degree of homology over most of the length of the gene or coding sequence e.g. histone and the α- and β-globin gene

families.

2- Members may have very low sequence homology but they posses one or more common protein domain e.g. the PAX and SOX gene families (Table 9.7).

3- Gene families are defined by functionally similar short protein motifs (these encode functionally-related protein with a DEAD (Asp-Glu-Ala-Asp) or the WD repeat (Fig. 9.9).

- Gene duplication events that give rise to multigene families also create pseudogenes and gene fragments.

- Pseudogenes are defective gene copies that contain multiple exons while gene fragments have only limited parts of the gene sequence (sometimes a single exon).

- Pseudogenes could be

(a) nonprocessed (e.g. Fig 9.8 and HLA gene family in Fig 9.10). May result from chromosmal locations that are unstable such as pericentromeric and subtelomeric regions. These regions are prone to recombination events that can result in duplicated gene segments being distributed to other chromosomal locations. Example of

pericentromeric rearrangenements is NF1 gene (Fig 9.11A) and subteolmeric rearrangements is polycystic kidney disease gene PKD1 (Fig 9.11B)

(b) processed via retrotransposition by cellular reverse transcriptase

(Fig. 9.12, Table 9.8)

RNA Genes

• Fig 9.13 shows the functional diversity of human ncRNA (noncoding RNA).

• Table 9.9 is a compilation of all the major classes of ncRNA

- More than a 1000 human genes, mostly within large gene clusters, encode rRNA or tRNA

- Ribosomal RNA genes:

- Two mitochondrial rRNA molecules (12S & 16S)

- Four types of cytoplasmic rRNA, 3 associated with the large ribosome subunit (28S, 5.8S, & 5S) and one with the small ribosome subunit (18S)

- The 5S occur in small gene clusters, the largest cluster is 16 genes on 1q42 close to the telomere.

- The 28S, 5.8S, & 18S rRNA are encoded by a single multigenic transcription unit that is tandemly repeated to form megabse- sized ribosomal DNA arrays (~30-40 tandem repeats or ~100 rRNA genes) on the short arms of each of the acrocentric

chromosomes 13, 14, 15, 21, & 22.

- Transfer RNA genes:

- 22 tRNA genes make 22 different tRNA molecules.

- Nuclear genome has 516 tRNA genes, classified into 49 families based on codon specificity, that make cytoplasmic tRNA .

- Amino acid frequency doesn’t correlate with the number tRNA genes. E.g. 30 tRNAs specify the rare cysteine (2.25% of all amino acids in

human proteins) but only 21 tRNA genes specify the more abundant proline (6.10% of total).

- More than half the tRNA genes (273 out of 516) reside in chromosome 6 (many clustered in a 4 Mb region) or 1. 18 of the 30 Cys tRNAs are found in a 0.5 Mb stretch of chromosome 7.

• Dispersed gene families make various small nuclear RNAs that facilitate general gene expression:

- Various families of small RNA molecules (60-360 nucleotides long) play a role in assisting general gene expression, mostly at the level of post-transcriptional processing.

There are 3 types:

(i) small nuclear RNAs (snRNAs) are U-rich and bind to various proteins to function as ribonucleoproteins (snRNPs).

(ii) snRNA that are involved in post-transcriptional processing of rRNA precursors in the nucleolus were re-classified as small nucleolar RNAs (snoRNAs).

(iii) Resemble snoRNAs but are confined coiled bodies (discrete structures in the nucleus that are involved with the maturation of SnRNPs) and are called Cajal body RNAs (scaRNAs).